Glutamate Transporter Research Articles

Unraveling the mechanistic interplay of mediators orchestrating the neuroprotective potential of harmine.

Neurodegenerative diseases (NDDs) encompass a range of conditions characterized by the specific dysfunction and continual decline of neurons, glial cells, and neural networks within the brain and spinal cord. The majority of NDDs exhibit similar underlying causes, including oxidative stress, neuroinflammation, and malfunctioning of mitochondria. Elevated levels of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), alongside decreased expression of brain-derived neurotrophic factor (BDNF) and glutamate transporter subtype 1 (GLT-1), constitute significant factors contributing to the pathogenesis of NDDs. Additionally, the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene has emerged as a significant target for the treatment of NDDs at the preclinical level. It significantly contributes to developmental brain defects, early onset neurodegeneration, neuronal loss, and dementia in Down syndrome. Moreover, an impaired ubiquitin-proteosome system (UPS) also plays a pathological role in NDDs. Malfunctioning of UPS leads to abnormal protein buildup or aggregation of α-synuclein. α-Synuclein is a highly soluble unfolded protein that accumulates in Lewy bodies and Lewy neurites in Parkinson's disease and other synucleinopathies. Recent research highlights the promising potential of natural products in combating NDDs relative to conventional therapies. Alkaloids have emerged as promising candidates in the fight against NDDs. Harmine is a tricyclic β-carboline alkaloid (harmala alkaloid) with one indole nucleus and a six-membered pyrrole ring. It is extracted from Banisteria caapi and Peganum harmala L. and exhibits diverse pharmacological properties, encompassing neuroprotective, antioxidant, anti-inflammatory, antidepressant, etc. Harmine has been reported to mediate its neuroprotective via reducing the level of inflammatory mediators, NADPH oxidase, AChE, BChE and reactive oxygen species (ROS). Whereas, it has been observed to increase the levels of BDNF, GLT-1 and anti-oxidant enzymes, along with protein kinase-A (PKA)-mediated UPS activation. This review aims to discuss the mechanistic interplay of various mediators involved in the neuroprotective effect of harmine.

Gypenoside XVII Reduces Synaptic Glutamate Release and Protects against Excitotoxic Injury in Rats.

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 μM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Oxidative stress plays an important role in the central regulatory mechanism of orofacial hyperalgesia under low estrogen conditions

Hyperalgesia occurs in the orofacial region of rats when estrogen levels are low, although the specific mechanism needs to be investigated further. Furthermore, oxidative stress plays an important role in the transmission of pain signals. This study aimed to explore the role of oxidative stress in orofacial hyperalgesia under low estrogen conditions. We firstly found an imbalance between oxidative and antioxidant capacity within the spinal trigeminal subnucleus caudalis (SP5C) of rats after ovariectomy (OVX), resulting in oxidative stress and then a decrease in the orofacial pain threshold. To investigate the mechanism by which oxidative stress occurs, we used virus as a tool to silence or overexpress the excitatory amino acid transporter 3 (EAAT3) gene. Further investigation revealed that the regulation of glutathione (GSH) and reactive oxygen species (ROS) can be achieved by regulating EAAT3, which in turn impacts the occurrence of oxidative stress. In summary, our findings suggest that reduced expression of EAAT3 within the SP5C of rats in the low estrogen state may decrease GSH content and increase ROS levels, resulting in oxidative stress and ultimately lead to orofacial hyperalgesia. This suggests that antioxidants could be a potential therapeutic direction for orofacial hyperalgesia under low estrogen conditions, though more research is needed to understand its mechanism.

Modulation of contextual fear acquisition and extinction by acute and chronic relaxin-3 receptor (RXFP3) activation in the rat retrosplenial cortex

The retrosplenial cortex (RSC) plays a central role in processing contextual fear conditioning. In addition to corticocortical and thalamocortical projections, the RSC receives subcortical inputs, including a substantial projection from the nucleus incertus in the pontine tegmentum. This GABAergic projection contains the neuropeptide, relaxin-3 (RLN3), which inhibits target neurons via its Gi/o-protein-coupled receptor, RXFP3. To assess this peptidergic system role in contextual fear conditioning, we bilaterally injected the RSC of adult rats with an adeno-associated-virus (AAV), expressing the chimeric RXFP3 agonist R3/I5 or a control AAV, and subjected them to contextual fear conditioning. The R3/I5 injected rats did not display any major differences to control-injected and naïve rats but displayed a significantly delayed extinction. Subsequently, we employed acute bilateral injections of the specific RXFP3 agonist peptide, RXFP3-Analogue 2 (A2), into RSC. While the administration of A2 before each extinction trial had no impact on the extinction process, treatment with A2 before each acquisition trial resulted in delayed extinction. In related anatomical studies, we detected an enrichment of RLN3-immunoreactive nerve fibers in deep layers of the RSC, and a higher level of co-localization of RXFP3 mRNA with vesicular GABA transporter (vGAT) mRNA than with vesicular glutamate transporter-1 (vGLUT1) mRNA across the RSC, consistent with an effect of RLN3/RXFP3 signalling on the intrinsic, inhibitory circuits within the RSC. These findings suggest that contextual conditioning processes in the RSC involve, in part, RLN3 afferent modulation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process.

Synaptic Profile and Oxidative Stress Associated with Amyloid-β in the Hippocampus

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.

Functional Impact of nTS Glutamate Stress on Respiration in an Alzheimer’s Disease Model

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.

Mechanisms Underlying Long-Term Facilitation in the Carotid Body

Glutamate and γ-aminobutyric acid (GABA) are major modulators of excitatory and inhibitory transmission in the mammalian brain. Glutamate transmission is critical for neural plasticity, learning and memory, whereas GABA plays a fundamental role in regulating neuronal excitability and facilitating the generation of neural oscillations. Our data indicates that both glutamate and GABA are released in the carotid body (CB) to modulates its response to hypoxia. Objectives: We set out to investigate whether repeated stimulation with glutamate and GABA induces lasting effects on CB afferent discharge. Hypothesis: We hypothesised that glutamate/GABA mediates long-term facilitation (LTF) of CB afferent activity. Methods: We mined high-throughput RNA sequence data and used immunohistochemistry to map components underlying neuroplasticity in the CB in Wistar and Spontaneously-hypertensive rats (SHR). CSN discharge was measured as a functional readout of CB function from an ex vivo arterially perfused carotid artery-CB preparation. Data: We catalogue the expression of glutamate receptors (R) and transporters in the rat and mouse CB. We found N-methyl-D-aspartic acid receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and transporters to be localised to the CB chemosensory cells. Targeted administration of 15 mM glutamate activated the carotid sinus nerve ( p=0.003). Repetitive [5 times, 2-minute duration, 5-minute intervals] application of 15 mM glutamate evoked LTF of CB afferent discharge as demonstrated by both a 12-fold increase in basal firing frequency (0.78 ± 0.37 vs 1232 ± 596 spikes 1s−1; p = 0.004) over 60 minutes ( p = 0.004) and amplification of the evoked response to chemical hypoxia (CN−; 1.23 μmol bolus) by 2-fold ( p = 0.009). The stimulation paradigm was specific as neither application of glutamate, 5 times for 1-minute at 5-minute intervals, nor a single glutamate exposure evoked LTF. In contrast, repeated application of 15mM GABA [3 times for 1-minute at 5-minute intervals] evoked long-term depression (LTD) of CB afferent activity as seen by a reduced basal discharge from 10.5 to 3.5 spikes 1s−1 ( p = 0.0004) and a 2-fold suppression of the response to chemical hypoxia ( p = 3.71 x 10−5). Summary of the data: Repetitive exposure to glutamate using a specific protocol increased basal discharge of CB afferent output akin to LTF and caused sensitisation of evoked responses to CN−. Repeated exposures of GABA depressed the CB basal discharge akin to LTD. A statement of the conclusions: It is conceivable that processes such as LTP and LTD operate in the CB to modulate the set point of peripheral chemoreceptor sensitivity and its state-dependent tonicity; the role of LTP in peripheral chemoreflex sensitisation in disease remains to be determined. Research was supported by the Health Research Council of New Zealand and the Sidney Taylor Trust. 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.

Chronic Vagotomy Blunts Cardiorespiratory Reflex Responses Through Pre- and Postsynaptic Effects in the Brainstem Nucleus Tractus Solitarii (nTS)

Sensory information from the heart, lungs, and gastrointestinal tract travels to the brain via afferents of the paired vagus nerves, where it is first integrated in the brainstem nTS that critically contributes to cardiorespiratory function. We have shown that reducing input to the nTS by unilaterally transecting the vagus nerve (vagotomy) induces astrocyte reactivity and microglial activation, and reduces sighs in hypoxia. However, the cardiorespiratory effects of vagotomy and contributing mechanisms are not well understood. We hypothesized that chronic vagotomy blunts cardiorespiratory reflex responses to vagal afferent stimulation via reduced nTS neuronal activity. Male Sprague-Dawley rats (6-wk) underwent right cervical vagotomy (or sham surgery) caudal to the nodose ganglion. After 7 days rats were anesthetized, ventilated, and instrumented to record mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively), which decreased in response to vagal afferent stimulation in sham animals. Vagotomy blunted these decreases in MAP, SSNA, and PhrNA compared to shams. In rats expressing the calcium indicator GCaMP8m in nTS neurons, vagotomy also reduced neuronal Ca2+ responses to afferent stimulation both in vivo (photometry) and in vitro (live slice imaging). We then hypothesized that these changes were due to altered signaling at the level of the tripartite synapse, where astrocytes enwrap the sensory afferent-nTS soma synapse. We found that there is likely a vagal afferent contribution to the altered reflex responses, as vagotomy reduced presynaptic Ca2+ responses in vagal afferent terminals to solitary tract stimulation in the brainstem slice. Glutamate agonists, which induce similar cardiorespiratory responses as vagal stimulation, were nanoinjected into the nTS to determine if there was a postsynaptic contribution to the blunted responses as well. Responses to glutamate injection were also blunted following vagotomy, indicating that astrocytic glutamate uptake or nTS soma glutamate receptor function may be altered. Blocking astrocytic excitatory amino acid transporters, which remove extracellular glutamate in the nTS induced similar baseline effects and did not restore cardiorespiratory responses to glutamate in vagotomy. However, nanoinjection of NMDA into the nTS to activate ionotropic glutamate receptors mimicked the blunted responses to glutamate following vagotomy, with no difference in responses to AMPA nanoinjection. Likewise, immunohistochemistry and Western blot showed that vagotomy reduced NMDA receptor subunit expression in the nTS. Together these results indicate that chronic vagotomy blunts cardiorespiratory responses to vagal afferent stimulation, likely through reduced presynaptic activation and decreased NMDA receptor expression and function. These studies will help improve our understanding of how afferent input induces neuroplasticity within the nTS, and the resulting impacts on cardiorespiratory function in health and disease. NIH HL132836, HL128454, HL98602, T32OD011126. 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.

Molecular organization of adrenergic and noradrenergic neurons in the mouse ventrolateral medulla

Catecholaminergic neurons in the ventrolateral medulla (VLM) regulate many important aspects of physiology, including the sympathetic control of blood pressure, glucose homeostasis, immune function, and the neuroendocrine responses to inflammation and stress. Previous studies have parsed catecholaminergic VLM neurons into distinct sub-populations based on neurochemical content, connectivity, and distinct response to the activation of autonomic reflexes. However, there is no consensus for the molecular organization of catecholaminergic neurons. In this study, we used high-throughput single nuclei RNA sequencing combined with anatomically- and genetically-targeted cell-sorting of spinally-projecting and catecholaminergic neurons to systematically characterize C1 and A1 neurons in the mouse VLM. Our results, based on an analysis of 894 catecholaminergic neurons derived from a dataset of 114,805 single-nuclei transcriptomes, indicate that the VLM contains 7 molecularly distinct subpopulations of catecholaminergic neurons, including 5 adrenergic subtypes and 2 noradrenergic subtypes identified by the expression of enzymes involved in the production of catecholamine ( Th, Dbh) and the vesicular transporters for glutamate ( Slc6a17) and noradrenaline ( Slc6a2). Neurons with projections to the spinal cord were present in 4 of 5 adrenergic cell clusters, and 1 noradrenergic cell cluster. We histologically verified two novel markers of the adrenergic ( Hk2) and noradrenergic ( Eya1) neurons using RNA fluorescent in situ hybridization. Hk2 expression was found primarily in Dbh+ neurons in the rostral VLM, including both bulbospinal and non-bulbospinal neurons, as well as majority of Pnmt+ neurons in the rostral VLM and C2/C3 regions. Eya1 co-localized with Dbh and Slc6a2 consistently in the caudal VLM, aligning with the location of the A1 neurons, whereas Eya1 signal was diffuse in the rostral VLM. Collectively, this shows that Hk2 expression is enriched in adrenergic neurons as judged by conventional criteria irrespective of the cell's location in the medulla, whereas Eya1 is enriched in brainstem noradrenergic neurons and provides a marker for A1 neurons. These data show molecularly distinct adrenergic and noradrenergic neurons are spatially intermixed in the VLM, and provides novel gene markers for distinct subtypes of catecholaminergic neurons that can be used to dissect the function in future studies. This work was supported by National Institute of Health (NIH) R01 HL148004 to SBGA; and R01 HL153916, Pathway to Stop Diabetes Initiator Award 1-18-INI-14, and UVA 3Cavaliers grant to JNC. 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.

17 β-Estradiol ameliorates cardiac parasympathetic dysfunction following myocardial infarction in male mice

Background: Vagal dysfunction after chronic myocardial infarction (MI) is associated with an increased risk of ventricular tachycardia (VT)/ventricular fibrillation (VF) and increased mortality in heart failure. The incidence and outcomes of VT/VF are influenced by sex, with women having a lower incidence of VT/VF associated with ischemic heart disease. However, the mechanisms behind these sex differences remain unclear and could be due to sex differences in autonomic remodeling post-MI that is mediated by estrogen. Hypotheses: We hypothesized that estrogen improves vagal function after MI by reducing oxidative stress in vagal sensory neurons and improving vagal afferent/efferent reflexes and function. Methods: Mice with expression of channelrhodopsin (ChR2) in specific sensory afferent neurons were created by crossing vesicular glutamate transporter 2 (VGlut2)-IRES-Cre mice with ChR2-EYFP mice, and their offspring (Vglut2-ChR2-EYFP) used for optogenetic stimulation studies. Sham surgery (n=6) or MI (n=6) was performed in two separate groups of male mice. A third group of male mice (n=8) underwent 17β-Estradiol (E2) pellet (0.5 milligrams/pellet) implantation followed by MI (E2+MI) after 2.5 weeks. Terminal studies were performed 2-3 weeks post-MI/sham procedures by placing a blue light (473 nm) laser probe over the left cervical vagus in vivo to stimulate vagal sensory neurons (20 Hz, 10 & 20 msec stimulations for 5 sec). Heart rate (HR) and blood pressure (BP) responses were measured in response to optogenetic stimulation. Plasma E2 and nodose ganglion 4-hydroxynaneol (4HNE) levels, a marker of lipid peroxidation, were evaluated. Results: In response to optogenetic vagal stimulation, MI had reduced reflex heart rate responses vs. sham animals, while E2+MI mice demonstrated significantly better responses vs. MI and similar responses to sham animals (20 Hz, 10 msec: sham -59 ± 3%, MI -24 ± 3%, E2+MI -58 ± 4%, MI vs. E2+MI, P < 0.001; 20 Hz, 20 msec: sham -59 ± 4%, MI -31 ± 3%, E2+MI -60 ± 3%, MI vs. E2+MI, P < 0.001). In addition, while BP responses post-MI were reduced vs. sham, E2+MI male mice also demonstrated greater BP responses to optogenetic stimulation (20 Hz, 10 msec: sham -28 ± 4%, MI -12 ± 1%, E2+MI -27± 1%, MI vs. E2+MI, P < 0.001; 20 Hz, 20 msec: sham -32 ± 4%, MI -13 ± 1%, E2+MI -27 ± 5%, MI vs. E2+MI, P < 0.001). 4HNE levels were increased in MI vs. sham animals, while E2+MI mice demonstrated significant reductions in 4HNE levels (Sham 0.17 ± 0.09 μg/protein, MI 0.77 ± 0.11 μg/protein, E2±MI 0.26 ± 0.06 μg/protein, MI vs. E2+MI, P < 0.001). Finally, plasma measurements confirmed higher levels of estrogen in E2-implanted mice (MI 48 ± 10 pg/mL, E2+MI 850 ± 170 pg/mL, MI vs. E2+MI, P < 0.001). Conclusions: MI causes pathological vagal remodeling that is reflected in decreased sensory vagal neurotransmission and increased oxidative stress in the vagal ganglia. Parasympathetic dysfunction and oxidative stress are mitigated by estrogen, suggesting that estrogen may be an important factor in mitigating the resulting pathological autonomic remodeling post-MI that predisposes to VT/VF. NIHR01HL148190. 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.

Immunohistochemical localization of proteins involved in exocytosis of glutamate from P2X3 purinoceptor-expressing subserosal afferent nerve endings in the rat gastric antrum

We previously reported the presence of P2X3 purinoceptors (P2X3)-expressing subserosal afferent nerve endings consisting of net- and basket-like nerve endings in the rat gastric antrum. These nerve endings may morphologically be vagal mechanoreceptors activated by antral peristalsis. The present study investigated immunoreactivities for vesicular glutamate transporter (VGLUT) 1 and VGLUT2 as well as exocytosis-related proteins, i.e., core components of the SNARE complex (SNAP25, Stx1, and VAMP2) and synaptotagmin-1 (Syt1), in whole-mount preparations of the rat gastric antrum using double immunofluorescence. VGLUT1 immunoreactivity was not detected, whereas VGLUT2 immunoreactivity was observed in P2X3-immunoreactive subserosal nerve endings composed of both net- and basket-like endings. In net-like nerve endings, intense VGLUT2 immunoreactivity was localized in polygonal bulges of reticular nerve fibers and peripheral axon terminals. Furthermore, intense immunoreactivities for SNAP25, Stx1, and VAMP2 were localized in net-like nerve endings. Intense immunoreactivities for VAMP2 and Syt1 were observed in VGLUT2-immunoreactive net-like nerve endings. In basket-like nerve endings, VGLUT2 immunoreactivity was localized in pleomorphic terminal structures and small bulges surrounding the subserosal ganglion, whereas immunoreactivities for SNAP25, Stx1, and VAMP2 were weak in these nerve endings. VGLUT2-immunoreactive basket-like nerve endings were weakly immunoreactive for VAMP2 and Syt1. These results suggest that subserosal afferent nerve endings release glutamate by exocytosis mainly from net-like nerve endings to modulate their mechanoreceptor function.

Specific biomarkers and neurons distribution of different brain regions in largemouth bass (Micropterus salmoides).

The brain regulates multiple physiological processes in fish. Despite this, knowledge about the basic structure and function of distinct brain regions in non-model fish species remains limited due to their diversity and the scarcity of common biomarkers. In the present study, four major brain parts, the telencephalon, diencephalon, mesencephalon and rhombencephalon, were isolated in largemouth bass, Micropterus salmoides. Within these parts, nine brain regions and 74 nuclei were further identified through morphological and cytoarchitectonic analysis. Transcriptome analysis revealed a total of 7153 region-highly expressed genes and 176 region-specifically expressed genes. Genes related to growth, reproduction, emotion, learning, and memory were significantly overexpressed in the olfactory bulb and telencephalon (OBT). Feeding and stress-related genes were in the hypothalamus (Hy). Visual system-related genes were predominantly enriched in the optic tectum (OT), while vision and hearing-related genes were widely expressed in the cerebellum (Ce) region. Sensory input and motor output-related genes were in the medulla oblongata (Mo). Osmoregulation, stress response, sleep/wake cycles, and reproduction-related genes were highly expressed in the remaining brain (RB). Three candidate marker genes were further identified for each brain regions, such as neuropeptide FF (npff) for OBT, pro-melanin-concentrating hormone (pmch) for Hy, vesicular inhibitory amino acid transporter (viaat) for OT, excitatory amino acid transporter 1 (eaat1) for Ce, peripherin (prph) for Mo, and isotocin neurophysin (itnp) for RB. Additionally, the distribution of seven neurotransmitter-type neurons and five types of non-neuronal cells across different brain regions were analyzed by examining the expression of their marker genes. Notably, marker genes for glutamatergic and GABAergic neurons showed the highest expression levels across all brain regions. Similarly, the marker gene for radial astrocytes exhibited high expression compared to other markers, while those for microglia were the least expressed. Overall, our results provide a comprehensive overview of the structural and functional characteristics of distinct brain regions in the largemouth bass, which offers a valuable resource for understanding the role of central nervous system in regulating physiological processes in teleost.

PREVENTION OF MERCURY-INDUCED EXCITOTOXICITY IN PRESYNAPTIC BRAIN NERVE TERMINALS WITH CARBON DOTS

Aim. Xenobiotic metal mercury is one of the major crucial pollutants of global public health concerns according to the World Health Organization assessment. Carbon-containing nanoparticles (CNPs) are promising in nanotechnology. CNPs were obtained by the combustion of citric acid and urea. Methods. The cortex nerve terminals isolated from Wistar rats were used in the experiments. [14C]glutamate uptake and release in the nerve terminals were monitored using a radiolabeled assay. Results. It was shown that HgCl2 starting from 5 µM caused a concentration-dependent increase in the extracellular L-[14C]glutamate level in nerve terminals resulted from weak functioning of glutamate transporter, and so significantly decreased L-[14C] glutamate uptake. Combined effects of Hg2+ and CNPs obtained by heating of citric acid and urea were analysed. CNPs were able to mitigate in an acute manner excitotoxic Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals by 37%, thereby being a provisional Hg2+ scavenger. Conclusion. Besides biotechnological implementation of data, developed approach can be applicable for monitoring capability of different particles and compounds to mitigate Hg2+ -mediated threat.

Transcriptional control of visual neural circuit development by GS homeobox 1.

As essential components of gene expression networks, transcription factors regulate neural circuit assembly. The homeobox transcription factor encoding gene, gs homeobox 1 (gsx1), is expressed in the developing visual system; however, no studies have examined its role in visual system formation. In zebrafish, retinal ganglion cell (RGC) axons that transmit visual information to the brain terminate in ten arborization fields (AFs) in the optic tectum (TeO), pretectum (Pr), and thalamus. Pretectal AFs (AF1-AF9) mediate distinct visual behaviors, yet we understand less about their development compared to AF10 in the TeO. Using gsx1 zebrafish mutants, immunohistochemistry, and transgenic lines, we observed that gsx1 is required for vesicular glutamate transporter, Tg(slc17a6b:DsRed), expression in the Pr, but not overall neuron number. gsx1 mutants have normal eye morphology, yet they exhibit impaired visual ability during prey capture. RGC axon volume in the gsx1 mutant Pr and TeO is reduced, and AF7 that is active during feeding is missing which is consistent with reduced hunting performance. Timed laser ablation of Tg(slc17a6b:DsRed)-positive cells reveals that they are necessary for AF7 formation. This work is the first to implicate gsx1 in establishing cell identity and functional neural circuits in the visual system.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic–cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Role of dopamine neurons in familiarity.

Dopamine neurons signal the salience of environmental stimuli and influence learning, although it is less clear if these neurons also determine the salience of memories. Ventral tegmental area (VTA) dopamine neurons increase their firing in the presence of new objects and reduce it upon repeated, inconsequential exposures, marking the shift from novelty to familiarity. This study investigates how dopamine neuron activity during repeated familiar object exposure affects an animal's preference for new objects in a subsequent novel object recognition (NOR) test. We hypothesize that a single familiarization session will not sufficiently lower dopamine activity, such that the memory of a familiar object remains salient, leading to equal exploration of familiar and novel objects and weaker NOR discrimination. In contrast, multiple familiarization sessions likely suppress dopamine activity more effectively, reducing the salience of the familiar object and enhancing subsequent novelty discrimination. Our experiments in mice indicated that multiple familiarization sessions reduce VTA dopamine neuron activation, as measured by c-Fos expression, and enhance novelty discrimination compared with a single familiarization session. Dopamine neurons that show responsiveness to novelty were primarily located in the paranigral nucleus of the VTA and expressed vesicular glutamate transporter 2 transcripts, marking them as dopamine-glutamate neurons. Chemogenetic inhibition of dopamine neurons during a single session paralleled the effects of multiple sessions, improving NOR. These findings suggest that a critical role of dopamine neurons during the transition from novelty to familiarity is to modulate the salience of an object's memory.

Synthesis and Structure-Activity Relationships for Glutamate Transporter Allosteric Modulators.

Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro. Herein, we report on the structure-activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Increased excitatory amino acid transporter 2 levels in basolateral amygdala astrocytes mediate chronic stress-induced anxiety-like behavior.

JOURNAL/nrgr/04.03/01300535-202506000-00024/figure1/v/2024-08-05T133530Z/r/image-tiff The conventional perception of astrocytes as mere supportive cells within the brain has recently been called into question by empirical evidence, which has revealed their active involvement in regulating brain function and encoding behaviors associated with emotions. Specifically, astrocytes in the basolateral amygdala have been found to play a role in the modulation of anxiety-like behaviors triggered by chronic stress. Nevertheless, the precise molecular mechanisms by which basolateral amygdala astrocytes regulate chronic stress-induced anxiety-like behaviors remain to be fully elucidated. In this study, we found that in a mouse model of anxiety triggered by unpredictable chronic mild stress, the expression of excitatory amino acid transporter 2 was upregulated in the basolateral amygdala. Interestingly, our findings indicate that the targeted knockdown of excitatory amino acid transporter 2 specifically within the basolateral amygdala astrocytes was able to rescue the anxiety-like behavior in mice subjected to stress. Furthermore, we found that the overexpression of excitatory amino acid transporter 2 in the basolateral amygdala, whether achieved through intracranial administration of excitatory amino acid transporter 2 agonists or through injection of excitatory amino acid transporter 2-overexpressing viruses with GfaABC1D promoters, evoked anxiety-like behavior in mice. Our single-nucleus RNA sequencing analysis further confirmed that chronic stress induced an upregulation of excitatory amino acid transporter 2 specifically in astrocytes in the basolateral amygdala. Moreover, through in vivo calcium signal recordings, we found that the frequency of calcium activity in the basolateral amygdala of mice subjected to chronic stress was higher compared with normal mice. After knocking down the expression of excitatory amino acid transporter 2 in the basolateral amygdala, the frequency of calcium activity was not significantly increased, and anxiety-like behavior was obviously mitigated. Additionally, administration of an excitatory amino acid transporter 2 inhibitor in the basolateral amygdala yielded a notable reduction in anxiety level among mice subjected to stress. These results suggest that basolateral amygdala astrocytic excitatory amino acid transporter 2 plays a role in in the regulation of unpredictable chronic mild stress-induced anxiety-like behavior by impacting the activity of local glutamatergic neurons, and targeting excitatory amino acid transporter 2 in the basolateral amygdala holds therapeutic promise for addressing anxiety disorders.

Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway.

Chemotherapy exposure has become a main cause of premature ovarian insufficiency (POI). This study aimed to evaluate the role and molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hUMSC-Exos) in ovarian function protection after chemotherapy. hUMSC-Exos were applied to cyclophosphamide-induced premature ovarian insufficiency mice and human ovarian granulosa tumor cells (KGN) to determine their effects on follicular development and granulosa cell apoptosis. Evaluation was done for iron ion and reactive oxygen species (ROS) production, lipid peroxidation levels, and changes in iron death-related molecules (nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Glutathione Peroxidase enzyme 4 (GPX4), and Solute carrier family 7 member 11 cystine glutamate transporter (SLC7A11; xCT)). Furthermore, rescue experiments using an Nrf2 inhibitor were performed to assess the therapeutic effects of hUMSC-Exos on granulosa cells. hUMSC-Exos promoted ovarian hormone levels and primary follicle development in POI mice and reduced granulosa cell apoptosis. After hUMSC-Exos treatment, the ROS production, free iron ions and lipid peroxidation levels of granulosa cells decreased, and the iron death marker proteins Nrf2, xCT and GPX4 also decreased. Furthermore, the Nrf2 inhibitor ML385 significantly attenuated the effects of hUMSC-Exos on granulosa cells. hUMSC-Exos inhibit ferroptosis and protect against CTX-induced ovarian damage and granulosa cell apoptosis through the Nrf2/GPX4 signaling pathway, revealing a novel mechanism of hUMSC-Exos in POI therapy.