The brain, despite comprising only 2% of body weight, consumes nearly 20% of the body’s oxygen, making it highly susceptible to hypoxic injury. Hypoxia triggers oxidative stress, cell death, and neurodegeneration, central to the pathophysiology of many neurological conditions like ischemic stroke, epilepsy, and Alzheimer’s disease. However, repeated mild hypoxia has been shown to activate endogenous neuroprotective mechanisms, enhancing cellular resilience. Intermittent hypoxic-hyperoxic training (IHHT) is emerging as a promising therapeutic approach to hypoxic injury, yet the mechanisms underlying its neuroprotective effects remain unclear. Proteomic studies aimed to identify these mechanisms are limited. This study focuses on identifying proteins and pathways altered by repeated mild hypoxia in the hippocampus, a brain region essential for memory and learning. Additionally, it aims to reveal sex-specific neuroprotective responses that could guide the development of targeted therapeutic strategies for brain injury. Eight-week-old male and female Sprague-Dawley rats from the ATSU KCOM facility were maintained under controlled conditions (12 hr light/dark cycle, 24°C, 40% humidity, water and food ad libitum). Rats were exposed to either room air (CTL, n = 3/sex) or to five 2-hour episodes of hypoxia (HX, 10% O 2 + 90% N 2 , 24 hours apart, n = 3/sex) using a BioSperix hypoxia chamber. 24 hours after the final exposure, hippocampi were dissected, flash frozen in liquid nitrogen, and stored at -80°C until sent for proteomic analysis (MetwareBio). Samples underwent protein extraction, enzymatic digestion, liquid chromatography, and high-resolution mass spectrometry. Data were analyzed using DIA-NN to identify differentially expressed proteins using a fold change threshold of ≥ 1.5 or ≤ 0.667 and a p-value of ≤ 0.05 (t-test), followed by bioinformatics-based annotation, enrichment, and visualization of affected pathways, protein interactions, subcellular localization, and expression patterns. Proteomics analysis identified a total of 11,333 proteins from 124,965 peptides. Quantitative results showed consistent abundance across biological replicates ensuring high reliability of the differential protein expression data. A total of 91 differentially expressed proteins were found in the male-CTL vs. male-HX group and a total of 52 were found in the female-CTL vs. female-HX group. Key findings include that males upregulate energy metabolism and synaptic plasticity proteins (e.g., PANK1, FXYD6) and females enhance blood-brain barrier integrity and lipid homeostasis proteins (e.g., ENHO, SCP2). Additionally, repeated mild hypoxia reduced Zdhhc8 expression in both male-HX and female-HX groups (compared to their CTLs), reducing neuronal palmitoylation activity and potentially impairing trafficking and signaling pathways. This study successfully used a proteomic approach to investigate the neuroprotective mechanisms activated in the rat hippocampus following repeated mild hypoxia exposure. Males primarily upregulated proteins associated with energy metabolism and synaptic plasticity, suggesting enhanced functional resilience. Females showed an enrichment of proteins related to blood-brain barrier integrity and lipid homeostasis, pointing toward structural and metabolic protection. Our data reveal significant sex-specific differences in the underlying neuroprotective pathways, emphasizing the need for sex-aware therapeutic approaches. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Ischemic stroke remains a leading cause of long-term disability and death worldwide. Current treatments restore cerebral blood flow but do not prevent reperfusion injury. Repeated exposure to mild hypoxia before stroke onset has shown in rats to reduce infarct volume and neuronal damage, suggesting activation of endogenous neuroprotective pathways. One such pathway is mediated by hypoxia-inducible factor 2α (HIF-2α), known to promote erythropoietin (EPO) and its receptor (EPOR), contributing to neuronal survival. Recent studies have highlighted cytokine receptor-like factor 3 (CRLF3) as an alternative EPOR mediating neuroprotection. This study aims to describe CRLF3 expression in the rat brain and whether repeated mild hypoxia upregulates HIF-2α and CRLF3 and compare these molecular responses in male versus female rats to guide sex-specific therapeutic development. Male and female Sprague-Dawley rats (8-10 weeks old) were randomly assigned to receive a daily 2-hour exposure of either normoxia (CTL, 21% O 2 /79% N 2 ; ~760 mmHg) or hypoxia (HX, 10% O 2 /90% N 2 ; ~760 mmHg) for either 3 (n = 6 males and 2 females/group) or 5 (n = 4/group/sex) consecutive days using a whole cage hypoxia chamber (BioSperix). Naïve rats were not exposed to the chambers (n = 2/group/sex). In a subset of males, we recorded breathing responses using plethysmography chambers (3 day-exposure; n = 5/group). Western blot was used to measure protein expression changes for HIF-2α and CRLF3 in forebrain sections. Data were analyzed using GraphPad Prism 9. First, we analyzed the physiological response of male rats to repeated mild hypoxia episodes. Rats increased minute ventilation from baseline during the 2-hour period of hypoxia, but responses did not differ between the 3 days (Two-way ANOVA; minute ventilation: ptime > 0.0001, pday = 0.782). This study is the first to analyze CRLF3 expression in rat brain tissue. Western blots of naïve tissue showed a single protein band at 55kDa, resembling the predicted molecular weight of 58kDa and expression was similar between both different brain regions and between male and female rats in each region. After 3 days of episodic hypoxia, neither HIF-2α nor CRLF3 expression differed between HX and CTL in either sex. After 5 days of episodic hypoxia HIF-2α and CRLF3 expression increased mildly in males, but not females, resulting in a significant difference between male-HX and female-HX groups for both proteins (One-way ANOVA; pHIF2a = 0.002; pCRLF3 = 0.0212). Physiological responses to hypoxia did not differ across the 3 days of episodic hypoxia, indicating that the mild hypoxia regimen did not induce a pathological stress response. CRLF3 is broadly expressed across rat brain in both sexes, suggesting its role in general cell function. The 3 days of episodic hypoxia was insufficient to alter their expression. However, 5 days mildly elevated HIF 2α and CRLF3 in males only. These findings suggest that HIF-2α-dependent mechanisms may contribute to CRLF3 regulation and that males exhibit greater molecular sensitivity to mild hypoxia than females. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Respiratory dysfunction is a prevalent comorbidity in Alzheimer's disease (AD), yet its underlying mechanisms are poorly understood. Using the Streptozotocin (STZ) -induced rat model of AD, which replicates respiratory dysfunction and brain pathologies observed in human AD, we analyzed how these impairments relate to central neurological integration within the peripheral chemoreflex. Our focus was on glutamatergic signaling at the synapse between peripheral chemoafferents and second-order neurons in the nucleus tractus solitarii (nTS), a critical brainstem center for respiratory control. Activating the peripheral chemoreflex with potassium cyanide (KCN) increased respiratory frequency. Response magnitudes to repeated KCN injections typically decreased over time, which was exacerbated in the STZ-AD group. Similarly, repeated glutamate nanoinjections into the caudal/commissural nTS caused a pronounced reduction of respiratory frequency responses in STZ-AD. Electrophysiological analysis of nTS neurons within the peripheral chemoreflex revealed increased network activity, enhanced excitatory postsynaptic currents evoked by solitary tract stimulation (TS-EPSCs), and elevated asynchronous glutamate release following high-frequency stimulation (aEPSCs). These data were consistent with molecular evidence for astrogliosis (elevated GFAP expression), reduced astrocytic glutamate uptake (decreased EAAT2 expression), and presynaptic calcium dysregulation (increased TRPV1 expression). Additionally, presynaptic metabotropic glutamate receptors (mGluR3) were downregulated, while postsynaptic ionotropic receptor expression remained unchanged. With high-frequency solitary tract stimulation, STZ-AD rats had greater frequency-dependent TS-EPSC depression than controls, mirroring the diminished respiratory responses to KCN and glutamate nanoinjections. Our findings connect neurophysiological and molecular changes at the first nTS synapse of the peripheral chemoreflex with impaired respiratory responses to hypoxia in the STZ-AD model.

Acute exposure to intermittent hypoxia (AIH) produces prolonged increases (long-term facilitation, LTF) in phrenic (PhrNA) and sympathetic (SNA) nerve activity (pLTF and sLTF, respectively) during non-hypoxic periods, and augments cardiorespiratory responses to hypoxia. We recently showed that neuronal activity in the nucleus tractus solitarii (nTS) is required for the induction and maintenance of LTF. However, the specific mechanisms involved were not determined. Because bouts of deoxygenation/reoxygenation produce reactive oxygen species and H2O2 contributes to plasticity in the nTS, we hypothesized that nTS H2O2 contributes to AIH-induced LTF and augmented hypoxic responses. We reduced H2O2 within the nTS acutely by nanoinjecting catalase or chronically by overexpressing catalase via an adenovirus vector. We then evaluated PhrNA and splanchnic SNA (SSNA) in animals subjected to AIH or time control. In control rats subjected to nTS nanoinjections of aCSF or overexpression of eGFP, AIH produced pLTF and sLTF, and augmented PhrNA responses to hypoxia. Reducing nTS H2O2 by either nTS nanoinjections or overexpression of catalase markedly attenuated the development of pLTF and sLTF. Augmented hypoxic responses due to AIH also were diminished. In contrast, after LTF had developed, nanoinjection of catalase had no effect on the magnitude of either PhrNA or SSNA although inhibiting nTS neuronal activity after LTF development reduced pLTF. The data indicate that nTS H2O2 is required for AIH-induced pLTF and sLTF, as well as augmentation of responses to hypoxia. Moreover, while nTS neuronal activity is essential to the maintenance of pLTF once developed, ongoing increases in H2O2 are not required.

Viscerosensory information travels to the brain via vagal afferents, where it is first integrated within the brainstem nucleus tractus solitarii (nTS), a critical contributor to cardiorespiratory function and site of neuroplasticity. We have shown that decreasing input to the nTS via unilateral vagus nerve transection (vagotomy) induces morphological changes in nTS glia and reduces sighs during hypoxia. The mechanisms behind post-vagotomy changes are not well understood. We hypothesized that chronic vagotomy alters cardiorespiratory responses to vagal afferent stimulation via blunted nTS neuronal activity. Male Sprague-Dawley rats (6 weeks old) underwent right cervical vagotomy caudal to the nodose ganglion, or sham surgery. After 1 week, rats were anaesthetized, ventilated and instrumented to measure mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively). Vagal afferent stimulation (2-50 Hz) decreased cardiorespiratory parameters and increased neuronal Ca2+ measured by in vivo photometry and in vitro slice imaging of nTS GCaMP8m. Vagotomy attenuated both these reflex and neuronal Ca2+ responses compared to shams. Vagotomy also reduced presynaptic Ca2+ responses to stimulation (Cal-520 imaging) in the nTS slice. The decrease in HR, SSNA and PhrNA due to nTS nanoinjection of exogenous glutamate also was tempered following vagotomy. This effect was not restored by blocking excitatory amino acid transporters. However, the blunted responses were mimicked by NMDA, not AMPA, nanoinjection and were associated with reduced NR1 subunits in the nTS. Altogether, these results demonstrate that vagotomy induces multiple changes within the nTS tripartite synapse that influence cardiorespiratory reflex responses to afferent stimulation. KEY POINTS: Multiple mechanisms within the nucleus tractus solitarii (nTS) contribute to functional changes following vagal nerve transection. Vagotomy results in reduced cardiorespiratory reflex responses to vagal afferent stimulation and nTS glutamate nanoinjection. Blunted responses occur via reduced presynaptic Ca2+ activation and attenuated NMDA receptor expression and function, leading to a reduction in nTS neuronal activation. These results provide insight into the control of autonomic and respiratory function, as well as the plasticity that can occur in response to nerve damage and cardiorespiratory disease.

Alzheimer’s disease (AD) is closely associated with obstructive sleep apnea (OSA). Such hypoxic insults trigger glutamate release of chemoafferents into the nucleus tractus solitarii (nTS), an important upstream center of the chemoreflex. Potential alterations of glutamate handling in the nTS may lead to the respiratory dysfunction seen in AD patients. Using the streptozotocin (STZ)-induced rat model — an effective proxy for human AD — we studied the functional consequences of nTS glutamate stress on respiration. STZ-AD was induced in 6-week-old male Sprague Dawley rats via intracerebroventricular injections of 2 - 2.25 mg/kg STZ. After two weeks, EMG recordings of diaphragm activity served as surrogate for respiratory responses to glutamate microinjections (20 nL of 40 mM) into the caudal nTS. In a subset of rats, chemoafferent terminals were labeled with the fluorescent tracer DiI to permit patch clamp recordings of identified 2nd order nTS neurons participating in the chemoreflex loop. Evoked glutamatergic excitatory postsynaptic currents (TS-EPSCs) were generated via tractus solitarii stimulation at frequencies (10 - 40 Hz) typical for afferent discharge during hypoxia. Acute glutamate injections into the caudal nTS increased respiratory frequency by 15 - 20 breaths per minute. The response magnitude was similar between CTL and STZ-AD animals. Excitatory stress with repeated glutamate injections (5 min. apart) evoked a reliable respiratory response in CTL that slowly declined to ~65% from its maximum over the course of 10 injections. In contrast, the decline of respiratory response in STZ-AD was more pronounced and occurred significantly earlier than in CTL, indicating altered glutamate handling in the caudal nTS of STZ-AD rats. Next, we used patch clamp electrophysiology to analyze increased glutamate release of chemoafferents onto 2nd order nTS neurons. High frequency stimulation of chemoafferents induced a successive depression of TS-EPSC amplitude in nTS neurons. TS-EPSC depression was significantly stronger in STZ-AD and may be due to altered glutamate handling at the synapse. Western Blot analysis of STZ-AD brainstem tissue including the nTS revealed a significant increase in glial fibrillary acidic protein (GFAP) expression and a decrease of excitatory amino acid transporters (EAATs) expression, suggesting astrocyte involvement in compromised nTS glutamate handling in STZ-AD. In summary, the STZ-AD model showed reduced respiratory responses to glutamate stress in the caudal nTS. The altered stress response may come from enhanced depression of TS-EPSC amplitude at nTS neurons in the chemoreflex. Altered astrocytic glutamate removal from the synaptic cleft may contribute to TS-EPSC depression. Together, altered glutamate handling in a respiratory brainstem center may contribute to OSA in AD patients. NIH R15AG065927 (TDO & DO), KCOM Biomedical Graduate Program (RKT &TDO), ATSU Research Support (SKRC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alzheimer’s Disease (AD) is a neurodegenerative disorder that leads to cognitive decline and memory loss. The neuropathology is mainly due to disturbances in neuronal circuits and protein aggregations of phosphorylated tau and Amyloid-β (Aβ). Other factors found in disease progression are oxidative stress, microglial activation, and decreased synaptic transmission. However, it is unknown whether these changes occur only in close association with Aβ plaques, or rather have broad occurrence throughout memory-related brain structures. Thus, we analyzed synaptic markers, glial cells, and oxidative stress in the memory associated CA1 hippocampal region of APP/PS1 transgenic mice. Specifically, we assessed these targets in the immediate vicinity and distant from Aβ plaques. Hippocampal sections (coronal, 30 μm thick) from 1-year-old APP/PS1 mice and their wild type controls (n = 6/group) were fluorescently labeled for synapse density (synaptophysin), vesicular glutamate transporter (vGLUT2), DNA oxidation (8-hydroxyguanosine, 8-OHG), microglia (Iba-1), astrocytes (GFAP), neurons (NeuN), and Aβ plaques (X-34). Images were taken in the CA1 region with a conventional fluorescent microscope and analyzed using ImageJ. Fluorescence intensity was then quantified for each group. Next, the intensity profile was plotted for each fluorophore across the width of the Aβ plaques. There were relatively few amyloid plaques in the hippocampal CA1 region of APP/PS1 mice when compared to other regions of the hippocampus and cerebral cortex. Within the CA1 region, and in absence of Aβ, there was no pronounced change in synaptic density, presynaptic glutamate transporters, neurons, DNA oxidation, astrocytes, or microglia when compared to wild-type control. However, fluorescent intensity of some targets changed markedly along the perimeter of occasionally occurring Aβ plaques in the CA1 region. While the intensity profile of synaptophysin declined gradually towards the center of Aβ plaques, oxidative stress and microglia increased in the immediate surroundings of Aβ. Unexpectedly, also the intensity of presynaptic glutamate transporter vGLUT2 increased towards the core of the Aβ plaque. In summary, our data showed increased vesicular glutamate transporter, microglia, and oxidative stress confined to the vicinity of Aβ plaques in APP/PS1 mice. Increased vGLUT2 levels due to Aβ have been previously associated with hyperexcitability in AD. The parallel or subsequent decrease in synaptophysin is viewed as a compensatory response to limit hyperactivity. Together, the present study reveals important insights about the dynamics of cellular components in the vicinity of Aβ, and that aggregates of Aβ differentially affect presynaptic components important for synaptic transmission. NIH R15AG065927 (TDO & DO), Truman State GIASR grant (TS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Obstructive sleep apnea is highly prevalent in Alzheimer's disease (AD). However, brainstem centers controlling respiration have received little attention in AD research, and mechanisms behind respiratory dysfunction in AD are not understood. The nucleus tractus solitarii (nTS) is an important brainstem center for respiratory control and chemoreflex function. Alterations of nTS integrity, like those shown in AD patients, likely affect neuronal processing and adequate control of breathing. We used the streptozotocin-induced rat model of AD (STZ-AD) to analyze cellular changes in the nTS that corroborate previously documented respiratory dysfunction. We used 2 common dosages of STZ (2 and 3 mg/kg STZ) for model induction and evaluated the early impact on cell populations in the nTS. The hippocampus served as control region to identify site-specific effects of STZ. There was significant atrophy in the caudal nTS of the 3 mg/kg STZ-AD group only, an area known to integrate che-moafferent information. Also, the hippocampus had significant atrophy with the highest STZ dosage tested. Both STZ-AD groups showed respiratory dysfunction along with multiple indices for astroglial and microglial acti-vation. These changes were primarily located in the caudal and intermediate nTS. While there was no change of astrocytes in the hippocampus, microglial activation was accompanied by a reduction in synaptic density. Together, our data demonstrate that STZ-AD induces site-specific effects on all major cell types, primarily in the caudal/intermediate nTS. Both STZ dosages used in this study produced a similar outcome and can be used for future studies examining the initial symptoms of STZ-AD.

Exposure to acute intermittent hypoxia (AIH) induces prolonged increases (long term facilitation, LTF) in phrenic and sympathetic nerve activity (PhrNA, SNA) under basal conditions, and enhanced respiratory and sympathetic responses to hypoxia. The mechanisms and neurocircuitry involved are not fully defined. We tested the hypothesis that the nucleus tractus solitarii (nTS) is vital to augmentation of hypoxic responses and the initiation and maintenance of elevated phrenic (p) and splanchnic sympathetic (s) LTF following AIH. nTS neuronal activity was inhibited by nanoinjection of the GABA(A) receptor agonist muscimol before AIH exposure or after development of AIH-induced LTF. AIH but not sustained hypoxia induced pLTF and sLTF with maintained respiratory modulation of SSNA. nTS muscimol before AIH increased baseline SSNA with minor effects on PhrNA. nTS inhibition also markedly blunted hypoxic PhrNA and SSNA responses, and prevented altered sympathorespiratory coupling during hypoxia. Inhibiting nTS neuronal activity before AIH exposure also prevented the development of pLTF during AIH and the elevated SSNA after muscimol did not increase further during or following AIH exposure. Furthermore, nTS neuronal inhibition after the development of AIH-induced LTF substantially reversed but did not eliminate the facilitation of PhrNA. Together these findings demonstrate that mechanisms within the nTS are critical for initiation of pLTF during AIH. Moreover, ongoing nTS neuronal activity is required for full expression of sustained elevations in PhrNA following exposure to AIH although other regions likely also are important. Together, the data indicate that AIH-induced alterations within the nTS contribute to both the development and maintenance of pLTF.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease affecting more than 50 million individuals worldwide. Sporadic AD comprises 95% of all cases and is most commonly known for its hallmark symptoms of cognitive decline and memory degradation. In addition, respiratory dysfunction is found in up to 80% of AD patients and often manifests as sleep-disordered breathing. In the early stages of AD, increased reactive oxygen species cause widespread oxidative stress, which can damage cellular components and severely impact neuronal activity. While the respiratory dysfunction and oxidative stress is well known, there is little known about the underlying mechanisms and their potential intersection in AD patients. This study used the Streptozotocin (STZ)-AD rat model, which is the only well-established model mimicking sporadic AD with its multifactorial presentations including AD hallmark symptoms and respiratory dysfunction. Two weeks after induction of the STZ-AD rat model (2 mg/kg STZ), we analyzed targets of oxidative stress in a critical respiratory control area in the brainstem, the nucleus Tractus Solitarii (nTS). Immunohistochemistry was used to fluorescently stain brainstem sections in order to analyze for oxidation of lipids (4-hydroxynonenal, 4-HNE), DNA (8-hydroxyguanine, 8-OHG), and protein sulfhydryl groups (dimedone-tagged sulfenic acid). While there was no pronounced lipid peroxidation, the STZ-AD group had significantly increased levels of DNA oxidation when compared to the control (CTL). In addition, sulfhydryl groups, which are present on many cellular proteins, also had a significantly increased baseline oxidation in the STZ-AD group when compared to CTL. Oxidation of these sulfhydryl groups is known to be responsible for altering ion channel function and thus neuronal activity. Reducing agent Dithiothreitol (DTT) was able to revert sulfhydryl oxidation in STZ-AD to its reduced form, while CTL rats did not change from baseline. On the other hand, addition of the oxidizing agent hydrogen peroxide increased sulfhydryl oxidation in CTL, but not in STZ-AD. Overall, our results show that STZ-AD causes increased oxidation throughout a key brainstem area of respiration. This oxidation also affects protein components important for neuronal activity, which could be an underlying cause for the respiratory dysfunction in AD. Funding sources include the Kirksville College of Osteopathic Medicine Biomedical Sciences Graduate Program and the National Institutes of Health (R15AG065927). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Respiratory dysfunction is commonly observed in Alzheimer's disease (AD) and likely due to neuronal alterations of the respiratory network in the brain. The streptozotocin (STZ) -induced AD model mimics respiratory problems seen in humans and exhibits neuronal hyperactivity in the nucleus tractus solitarii (nTS), the central integration site for respiratory afferents in the brainstem. This ongoing study looks at the changes of glutamate signaling within the chemoreflex axis as a possible mechanism for nTS hyperactivity and respiratory dysfunction in STZ-AD. AD was induced by intracerebroventricular injection of 2 mg/kg STZ in 6-week old male Sprague Dawley rats. After 14 days, caudal nTS neurons within close proximity of DiI-labeled chemoafferent terminals were recorded using the patch clamp technique. Electrical stimulation of the tractus solitarii (TS) generated glutamatergic excitatory postsynaptic currents (EPSCs) that were compared between experimental groups. Consistent with enhanced glutamatergic signaling, TS-EPSC amplitude was increased in STZ-AD. High TS stimulation frequency induced a time and frequency dependent depression that was similar between groups, indicating no change in presynaptic vesicle turnover or postsynaptic receptor desensitization. On the other hand, asynchronous EPSCs following high frequency stimulation seemed enhanced in STZ-AD, supporting an increased excitatory tone in the nTS. Immunohistochemical analysis of the caudal nTS revealed no change in neuron number but showed a reduced synaptic density in the AD model. Astrocyte number and morphology were unchanged between groups. However, microglia, which are able to influence glutamate-handling of astrocytes, showed an activated phenotype. Overall, our data show enhanced glutamatergic signaling in the chemoreflex axis contributing to nTS hyperactivity of the STZ-AD model. nTS hyperactivity may lead to the observed respiratory dysfunction in this model and potentially constitutes a mechanism for sleep disordered breathing in AD patients.

BACKGROUND:Alzheimer's disease (AD) patients frequently present with orthostatic hypotension. This inability to reflexively increase blood pressure on standing is a serious health concern and increases the risk of stroke and cardiovascular diseases.OBJECTIVE:Since there are no clear mechanisms for orthostatic hypotension in human AD, the present study assessed the autonomic changes that could explain this comorbidity in an AD animal model.METHODS:We used the established streptozotocin-induced rat model of AD (STZ-AD), which mimics many hallmark symptoms of sporadic AD in humans. Baroreflex responses were analyzed in anesthetized STZ-AD rats using femoral catheterization for blood pressure and heart rate, and autonomic activity was assessed using specific blockers and splanchnic sympathetic nerve recordings. Expression levels of autonomic receptors at the heart were examined using the western blot technique.RESULTS:Baroreflex function in STZ-AD showed a blunted heart rate (HR) response to low blood pressure challenges, and the maximal sympathetic nerve activity was reduced. Conversely, HR responses to high blood pressure were similar to control, indicating no change in parasympathetic nerve activity. Under resting conditions, autonomic blockade demonstrated a baseline shift to increased sympathetic tone in STZ-AD. Protein expression levels of beta-1 adrenergic receptor and muscarinic acetylcholine receptor M2 in the heart were unchanged.CONCLUSION:Our study provides the first data on the pathological influence of AD on baroreflex function, which primarily affected the sympathetic nervous system in STZ-AD. These results represent the first mechanisms that may correlate with the orthostatic hypotension in human AD.

The locus coeruleus (LC) is a pontine nucleus important for respiratory control and central chemoreception. It is affected in Alzheimer's disease (AD) and alteration of LC cell function may account for respiratory problems observed in AD patients. In the current study, we tested the electrophysiological properties and CO2/pH sensitivity of LC neurons in a model for AD. Sporadic AD was induced in rats by intracerebroventricular injection of 2 mg/kg streptozotocin (STZ), which induces behavioral and molecular impairments found in AD. LC neurons were recorded using the patch clamp technique and tested for responses to CO2 (10% CO2, pH = 7.0). The majority (~60%) of noradrenergic LC neurons in adult rats were inhibited by CO2 exposure as indicated by a significant decrease in action potential (AP) discharge to step depolarizations. The STZ-AD rat model had a greater sensitivity to CO2 than controls. The increased CO2-sensitivity was demonstrated by a significantly stronger inhibition of activity during hypercapnia that was in part due to hyperpolarization of the resting membrane potential. Reduction of AP discharge in both groups was generally accompanied by lower LC network activity, depolarized AP threshold, increased AP repolarization, and increased current through a subpopulation of voltage-gated K+ channels (KV). The latter was indicated by enhanced transient KV currents particularly in the STZ-AD group. Interestingly, steady-state KV currents were reduced under hypercapnia, a change that would favor enhanced AP discharge. However, the collective response of most LC neurons in adult rats, and particularly those in the STZ-AD group, was inhibited by CO2.

Introduction: Locus Coeruleus (LC) is an important chemosensitive nucleus and affected by neurodegenerative diseases like Alzheimer's disease (AD). LC dysfunction in AD may account for the respiratory problems observed in patients. Objective: To test the electrophysiological properties of LC neurons in a model for sporadic AD. Material and Methods: AD was induced in rats (6-7 weeks) by intracerebroventricular injection of streptozotocin (STZ; 2 mg/kg). 14 days following injection, LC neurons were recorded using the patch clamp technique and tested for CO2 chemosensitivity (10% CO2, pH = 7.0). Results: When exposure hypercapnic condition, most LC neurons (~60%) exhibited a blunted spike discharge to current injections in comparison to baseline responses. The minority of cells either increased spiking (~20%) or did not respond (~20%) to CO2. Within cells that were inhibited by CO2, current-evoked spike discharge at baseline condition had the same magnitude in control and STZ rats. Responses in both groups decreased significantly when exposed to 10% CO2 (bsl vs. 10% CO2: CTL, p=0.003, n=8 and STZ, p=0.001, n=9). In the STZ group, this CO2-induced decrease in spike discharge was more pronounced when compared to control (CTL vs. STZ, p=0.038), suggesting greater sensitivity to hypercapnia. There was no difference in resting membrane potential and input resistance (Ri, cell membrane resistance) between groups at baseline and CO2. However, although there was no difference between groups values, we found a significant decrease of Ri within the STZ group when exposed to hypercapnia (bsl, 126.5 ± 14.9 MΩ vs. 10% CO2, 98.4 ± 8.2 MΩ; p=0.01), indicating opening of ion channels. The current-voltage relationship of the cell membrane showed a significant CO2-induced decrease of the steady state current (bsl vs. 10% CO2: CTL, p=0.002, and STZ, p=0.001). The magnitude was similar in both groups. This result would paradoxically favor increased excitability in neurons. Analysis of action potential (AP) parameters (AP threshold, AP peak, upstroke slope, peak to anti-peak) also showed no difference between groups. However, within the STZ group spike threshold was significantly shifted to more positive potentials under increased CO2 (-39.6 ± 1.9 mV vs -34.8 ± 2.2 mV, p=0.01). This shift in spike threshold would explain the blunted spike discharge of LC neurons in the STZ group during hypercapnic conditions. Conclusions: In summary, our data suggest that the majority of LC neurons in adult rats are inhibitedunder CO2 exposure. Further, the STZ-treated group exhibits a greater sensitivity to CO2, likely due to an increased spike threshold and opening of additional, yet unidentified membrane channels. Decreased excitability of LC neurons may be an underlying mechanism for the breathing disturbances observed in patients with AD. Keywords: CO2,neurons,adult rats, locus coeruleus Neurons, streptozotocin-Induced model, Alzheimer's Disease

Alzheimer’s disease (AD) is associated with deterioration of memory and cognition due to changes in brain areas important for these functions. The majority of AD patients also suffer multiple episodes of breathing cessations (apneas) during sleep. AD‐related sleep apnea may be associated with the decline of brainstem centers that control breathing and chemoreflex function. The nucleus tractus solitarii (nTS) is a key region for respiratory control and any AD‐related deterioration of this nucleus critically impacts its function.In this study, we used the streptozotocin (STZ) ‐induced rat model for sporadic AD (6‐week old rats, N = 6–8 rats/group) and compared chemoreflex dysfunction to indices of morphological alterations in the nTS. Particularly, we compared data obtained two weeks following injections of either 2 mg/kg or 2× 1.5 mg/kg STZ. Peripheral chemoreflex function was tested with 10% O2 in a plethysmography chamber. While increased respiratory rate thirty minutes into hypoxia was significantly blunted with both STZ dosages, animals receiving 2 mg/kg STZ partially compensated with larger tidal volumes. Hence, minute ventilation (rate x volume) was only significantly decreased in animals that received 2× 1.5 mg/kg STZ. Consistent with previously published data analyzing memory dysfunction in this model, there was a trend for reduced overall hippocampal size for both STZ concentrations. Immunohistochemical analysis of neuronal density (Anti‐NeuN antibody) in the CA1 region showed no difference between both STZ concentrations and control. However, synaptic density (Anti‐Synaptophysin antibody) was significantly reduced in both STZ concentrations. The overall size of the caudal nTS (4 sections each 180 μm apart) in the brainstem exhibited a greater reduction in 2× 1.5 mg/kg STZ. NeuN density was not altered between STZ concentrations and control. Interestingly, despite the stronger decline of chemoreflex function and nTS size with 2× 1.5 mg/kg STZ, synaptic density in the nTS (calamus scriptorius area) yielded a significant reduction for 2 mg/kg STZ only.Our study shows that reduced nTS size and synaptic density may be underlying the early changes of chemoreflex dysfunction in this model of sporadic AD. 2× 1.5 mg/kg STZ seemed to have a greater impact on respiratory function and nTS size. However, the varying change in synaptic density of each STZ concentration may be due to regional differences, since nTS size at calamus scriptorius did not differ between groups. Future studies will incorporate analysis of more caudal and rostral sections.Support or Funding InformationTruScholar and MoLSAMP grant from Truman State University (TSU) to CMH, GIASR from TSU to MW, seed money from A.T. Still University to TDO

BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative disorder causing multiple autonomic conditions including cardiovascular impairments. The inability to maintain blood pressure via baroreflex regulation is up to 5.6 times more prevalent in AD and patients present most commonly with orthostatic hypotension. This study aimed to assess the influence of AD on autonomic regulation of blood pressure using the streptozotocin (STZ)‐induced AD rat model, which mimics many hallmark symptoms of sporadic AD in humans.MethodsMale Sprague‐Dawley rats (345 ± 18 g) were randomly divided into two groups and subjected to intracerebroventricular injection of either vehicle (citrate buffer, CTL) or 2 mg/kg STZ in citrate buffer. After two weeks, memory performance (Morris water maze, MWM), baroreflex function (via femoral cannulation), and heart rate variability (HRV) were evaluated.ResultsMWM verified memory impairment in STZ‐treated rats when compared to CTL (p≤0.01). Analysis of baroreflex function in anesthetized rats revealed a significant impairment in the STZ group when compared to CTL. Particularly, STZ‐treated rats were unable to increase compensatory HR at lower blood pressures (at 50 mmHg: CTL, n=5, 369 ± 13 bpm vs. STZ, n=7, 315 ± 14 bpm; p≤0.05). STZ rats also had a lower baroreflex gain indicating reduced sensitivity to blood pressure fluctuations (p≤0.001). Additionally, recordings of splanchnic sympathetic nerve activity (SSNA) in a subset of rats identified reduced nerve activity during low blood pressure in the STZ group (∫SSNA at 50 mmHg: CTL, n=3, 0.3 ± 0.03 mV.s vs. STZ, n=3, 0.17 ± 0.02 mV.s; p≤0.05). There was no difference between groups at higher blood pressures (150 mmHg). Within the STZ group, HRV was significantly increased only during PE infusion (SDNN, p≤0.05; SD1, p≤0.05; SD2, p≤0.05). Interestingly, the change in HR following blockage of sympathetic or parasympathetic input was not different between groups. Also intrinsic HR and the blood pressure change to ganglionic blockage were similar between STZ and CTL rats.ConclusionsOur study provides the first insight into the pathological influence of AD on baroreflex function. We showed that STZ rats exhibit lower HR and reduced sympathetic output in response to low blood pressure. These results provide the basis for identifying the mechanism behind orthostatic hypotension in AD patients.Support or Funding InformationATSU biomedical grant (JCE) and seed money (TDO)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Patients with Alzheimer's disease (AD) develop pathological changes in the Locus Coeruleus (LC), an important pontine nucleus involved in respiratory control and central chemoreception. In addition, AD patients have breathing disturbances that may be related to LC dysfunction. Here, we tested the electrophysiological properties of LC neurons in a model for AD. Sporadic AD was induced in rats (6–7 weeks) by intracerebroventricular injection of streptozotocin (STZ; 2 mg/kg). After 14 days following injection, LC neurons were recorded using the patch clamp technique and tested for CO 2 chemosensitivity (10% CO 2 , pH = 7.0). Hypercapnic exposure lowered current‐evoked spike discharge in the majority of LC neurons (~60%) when compared to baseline responses. The remaining cells either increased spiking (~20%) or did not respond (~20%) to CO 2 . Within the cell group that was inhibited by hypercapnia, baseline spike discharge to current injection was similar between control and STZ rats. These responses decreased significantly in both groups when exposed to 10% CO 2 (bsl vs. 10% CO 2 : CTL, p=0.003, n=8 and STZ, p=0.001, n=9). The CO 2 ‐induced decrease in spike discharge of the STZ group was significantly lower when compared to control (CTL vs. STZ, p=0.038), indicating greater sensitivity to hypercapnia. However, we observed no difference in resting membrane potential and input resistance (R i , resistance across the cell membrane) between groups at baseline and CO 2 . Despite lack of difference between groups, within the STZ group R i decreased significantly with hypercapnia (bsl, 126.5 ± 14.9 MΩ vs. 10% CO 2 , 98.4 ± 8.2 MΩ; p=0.01), indicating ion channel opening when exposed to CO 2 . Analysis of the current‐voltage relationship only showed a significant decrease in the steady state current under CO 2 (bsl vs. 10% CO 2 : CTL, p=0.002, and STZ, p=0.001,), which had a similar magnitude in both groups. Close analysis of action potential (AP) shape (spike threshold, peak, slope, peak to anti‐peak) showed no difference between groups. Within the STZ rats, however, spike threshold was significantly shifted to more positive potentials when exposed to CO 2 (−39.6 ± 1.9 mV vs −34.8 ± 2.2 mV, p=0.01). This shift in spike threshold likely contributes to the pronounced decrease in spike discharge in the STZ group during hypercapnic conditions. In summary, our data suggest that the majority of LC neurons in adult rats are inhibited by CO 2 . Further, the STZ‐treated group exhibits a greater sensitivity to CO 2 , possibly due to an increased spike threshold and opening of additional, yet unidentified membrane channels. A decreased excitability of LC neurons may play a role in the respiratory dysfunction observed in patients with AD. Support or Funding Information FAPESP 2017/21750‐9 (MCV); ATSU seed money (TDO) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Insects with ears process sounds and respond to conspecific signals or predator cues. Axons of auditory sensory cells terminate in mechanosensory neuropils from which auditory interneurons project into (brain-) areas to prepare response behaviors. In the prothoracic ganglion of a bush-cricket, a cluster of local DUM (dorsal unpaired median) neurons has recently been described and constitutes a filter bank for carrier frequency. Here, we demonstrate that these neurons also constitute a filter bank for temporal patterns. The majority of DUM neurons showed pronounced phasic-tonic responses. The transitions from phasic to tonic activation had different time constants in different DUM neurons. Time constants of the membrane potential were shorter in most DUM neurons than in auditory sensory neurons. Patterned stimuli with known behavioral relevance evoked a broad range of responses in DUM neurons: low-pass, band-pass, and high-pass characteristics were encountered. Temporal and carrier frequency processing were not correlated. Those DUM neurons producing action potentials showed divergent processing of temporal patterns when the graded potential or the spiking was analyzed separately. The extent of membrane potential fluctuations mimicking the patterned stimuli was different between otherwise similarly responding neurons. Different kinds of inhibition were apparent and their relevance for temporal processing is discussed.

Major depressive disorder (MDD) is arguably the largest contributor to the global disease and disability burden, but very few treatment options exist for juvenile MDD patients. Ghrelin is the principal hunger-stimulating peptide, and it has also been shown to reduce depressive-like symptoms in adult rodents. We examined the effects of intracerebroventricular (icv) injection of ghrelin on depressive-like behavior. Moreover, we determined whether ghrelin increased neurogenesis in the hippocampus. Ghrelin (0.2-nM, 0.5-nM, and 1.0-nM) was administered acutely by icv injection to juvenile rats to determine the most effective dose (0.5-nM) by a validated feeding behavior test and using the forced swim test (FST) as an indicator of depressive-like behavior. 0.5-nM ghrelin was then administered icv against an artificial cerebrospinal fluid (aCSF) vehicle control to determine behavioral changes in the tail suspension test (TST) as an indicator of depressive-like behavior. Neurogenesis was investigated using a mitogenic paradigm, as well as a neurogenic paradigm to assess whether ghrelin altered neurogenesis. Newborn hippocampal cells were marked using 5'-bromo-2'-deoxyuridine (BrdU) administered intraperitoneally (ip) at either the end or the beginning of the experiment for the mitogenic and neurogenic paradigms, respectively. We found that ghrelin administration increased immobility time in the TST. Treatment with ghrelin did not change mitogenesis or neurogenesis. These results suggest that ghrelin administration does not have an antidepressant effect in juvenile rats. In contrast to adult rodents, ghrelin increases depressive-like behavior in male juvenile rats. These results highlight the need to better delineate differences in the neuropharmacology of depressive-like behavior between juvenile and adult rodents.