Ionotropic Glutamate Receptor Antagonists Research Articles

Inactivation of the paraventricular nucleus attenuates the cardiogenic sympathetic afferent reflex in the spontaneously hypertensive rat.

Myocardial ischemia causes the release of bradykinin, which stimulates cardiac afferents, causing sympathetic excitation and chest pain. Glutamatergic activation of the paraventricular hypothalamic nucleus (PVN) in the spontaneously hypertensive rat (SHR) drives elevated basal sympathetic activity. Thus, we tested the hypothesis that inactivation of the PVN attenuates the elevated reflex response to epicardial bradykinin in the SHR and that ionotropic PVN glutamate receptors mediate the elevated reflex. We recorded the arterial pressure and renal sympathetic nerve activity (RSNA) response to epicardial bradykinin application in anesthetized SHR and Wistar Kyoto (WKY) rats before and after PVN microinjection of GABA A agonist muscimol or ionotropic glutamate receptor antagonist kynurenic acid. Muscimol significantly decreased the arterial pressure response to bradykinin from 180.4 ± 5.8 to 119.5 ± 6.9 mmHg in the SHR and from 111.8 ± 7.0 to 84.2 ± 8.3 mmHg in the WKY and the RSNA response from 186.2 ± 7.1 to 142.7 ± 7.3% of baseline in the SHR and from 201.0 ± 11.5 to 160.2 ± 9.3% of baseline in the WKY. Kynurenic acid significantly decreased the arterial pressure response in the SHR from 164.5 ± 5.0 to 126.2 ± 7.7 mmHg and the RSNA response from 189.9 ± 13.7to 168.5 ± 12.7% of baseline but had no effect in the WKY. These results suggest that tonic PVN activity is critical for the full manifestation of the CSAR in both the WKY and SHR. Glutamatergic PVN activity contributes to the augmented CSAR observed in the SHR.

FRI275 Glutamate Receptor Agonists Stimulate Kisspeptin Neurons In The Arcuate Nucleus Of The Hypothalamus

Abstract Disclosure: R. Bearss: None. R. Piet: None. KNDy neurons co-express kisspeptin, neurokinin B (NKB), dynorphin A (Dyn A), and glutamate. These neurons, located in the arcuate nucleus of the hypothalamus, drive pulsatile gonadotropin releasing hormone (GnRH) secretion. This eventually promotes pulsatile gonadotropin hormone release from the anterior pituitary, important for fertility in both sexes. The current hypothesized mechanism for pulse generation involves KNDy neuron interconnectivity and reciprocal regulation through the combined actions of NKB, DynA and glutamate to coordinate KNDy neuron activity. There is evidence that KNDy neurons express NKB, Dyn A and glutamate receptors and that NKB stimulates KNDy neuron electrical activity while Dyn A suppresses it. There is also evidence that ionotropic glutamate receptor antagonists can inhibit KNDy neuron coordinated activity and pulsatile gonadotropin hormone release, indicating that glutamatergic neurotransmission plays a significant role in activity coordination. The purpose of the following experiments was to investigate the response of KNDy neurons to varied glutamate receptor activation. We used calcium imaging to monitor the activity of KNDy neurons in brain slices. We crossed Kiss1-Cre mice—expressing Cre recombinase (Cre) in kisspeptin neurons—with Cre-dependent GCaMP6f mice to generate Kiss1-Cre x GCaMP6f mice that express GCaMP6f in kisspeptin neurons. GCaMP6f fluorescence increases when binding to intracellular calcium and can, thus, be used as an inferential marker of electrical activity. Coronal brain slices (200 µm thickness) obtained from male Kiss1-cre x GCaMP6f mice and containing the arcuate nucleus were placed in a recording chamber under an epifluorescence microscope. KNDy neurons were exposed for 2 minutes to a bath application of either the ionotropic glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 10µM), the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA, 50µM), or the group 1 metabotropic glutamate receptor (mGluR) agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 50µM). AMPA, NMDA, and DHPG increased KNDy neuron fluorescence by 7.8±0.5% (n=113 in 7 slices from 6 mice), 7.5±0.5% (n=154 in 6 slices from 5 mice), and 6.1±0.7% (n=159 in 7 slices from 5 mice) respectively. All cells that were excited by NMDA or DHPG were also excited by AMPA. These experiments provide evidence that activation of ionotropic AMPA and NMDA receptors as well as group 1 mGluRs stimulate KNDy neuron activity. Further investigation into the precise role of individual glutamate receptor subtypes on KNDy neuron coordinated activity is needed. Presentation: Friday, June 16, 2023

Convenient One-Pot Synthesis of Kynurenic Acid Ethyl Ester and Exploration to Direct Synthesis of Neuroprotective Kynurenic Acid and Amide Derivatives.

Kynurenic acid (KYNA) is an endogenous molecule, which is a non-selective antagonist of ionotropic glutamate receptors and has been found to have neuroprotective activity. A supplement of KYNA may be applicable for the treatment of neurodegenerative disease, but it does not cross the blood-brain barrier due to its polar nature. Therefore, its different esters and amide derivatives were explored as a prodrug, which can cross blood-brain barrier and transform into KYNA in situ. However, many esters and amide derivatives of KYNA are synthesized via coupling reaction or multi-step synthesis using different organic or metallic catalysts. Herein, we developed a novel one-pot, catalyst-free, convenient synthesis of KYNA ethyl esters using aniline and diethyl acetylene dicarboxylate in DMF under heating. We also explored the synthesis of KYNA and KYNA amide derivative in a simple manner with overall good yields via hydrolysis and condensation, respectively.

Complex Regional Pain Syndrome: Clinic, Diagnosis, Treatment

The scientific work highlights the main issues of complex regional pain syndrome. A brief historical background on the treatment and diagnosis of this disease is given. The article provides a definition, a brief description of risk factors, epidemiology, pathophysiology, clinical picture of complex regional pain syndrome, outlines the basic principles of treatment of this disease. Timely diagnosis and early treatment of the disease are of great importance. All patients need medication therapy, physiotherapy, physical therapy, rehabilitation, psychological support throughout the disease. Drug therapy is represented by a number of groups of drugs: glucocorticosteroids, NSAIDs, analgesics, antidepressants, anticonvulsants, calcitonin, muscle relaxants, bisphosphonates, antagonists of ionotropic glutamate receptors. Interventional and surgical treatment is also assigned a role.

Characterization of Non-Specific Uptake and Retention Mechanisms of [177Lu]Lu-PSMA-617 in the Salivary Glands.

The radionuclide therapy [177Lu]Lu-PSMA-617 was recently FDA-approved for treatment of metastatic castration-resistant prostate cancer. Salivary gland toxicity is currently considered as the main dose-limiting side effect. However, its uptake and retention mechanisms in the salivary glands remain elusive. Therefore, our aim was to elucidate the uptake patterns of [177Lu]Lu-PSMA-617 in salivary gland tissue and cells by conducting cellular binding and autoradiography experiments. Briefly, A-253 and PC3-PIP cells, and mouse kidney and pig salivary gland tissue, were incubated with 5 nM [177Lu]Lu-PSMA-617 to characterize its binding. Additionally, [177Lu]Lu-PSMA-617 was co-incubated with monosodium glutamate, ionotropic or metabotropic glutamate receptor antagonists. Low, non-specific binding was observed in salivary gland cells and tissues. Monosodium glutamate was able to decrease [177Lu]Lu-PSMA-617 in PC3-PIP cells, mouse kidney and pig salivary gland tissue. Kynurenic acid (ionotropic antagonist) decreased the binding of [177Lu]Lu-PSMA-617 to 29.2 ± 20.6% and 63.4 ± 15.4%, respectively, with similar effects observed on tissues. (RS)-MCPG (metabotropic antagonist) was able to decrease the [177Lu]Lu-PSMA-617 binding on A-253 cells to 68.2 ± 16.8% and pig salivary gland tissue to 53.1 ± 36.8%. To conclude, we showed that the non-specific binding on [177Lu]Lu-PSMA-617 could be reduced by monosodium glutamate, kynurenic acid and (RS)-MCPG.

The Basolateral Amygdala Sends a Mixed (GABAergic and Glutamatergic) Projection to the Mediodorsal Thalamic Nucleus.

The medial prefrontal cortex receives converging inputs from the mediodorsal thalamic nucleus (MD) and basolateral amygdala (BLA). Although many studies reported that the BLA also projects to MD, there is conflicting evidence regarding this projection, with some data suggesting that it originates from GABAergic or glutamatergic neurons. Therefore, the present study aimed to determine the neurotransmitter used by MD-projecting BLA cells in male and female rats. We first examined whether BLA cells retrogradely labeled by Fast Blue infusions in MD are immunopositive for multiple established markers of BLA interneurons. A minority of MD-projecting BLA cells expressed somatostatin (∼22%) or calretinin (∼11%) but not other interneuronal markers, suggesting that BLA neurons projecting to MD not only include glutamatergic cells, but also long-range GABAergic neurons. Second, we examined the responses of MD cells to optogenetic activation of BLA axons using whole-cell recordings in vitro Consistent with our immunohistochemical findings, among responsive MD cells, light stimuli typically elicited isolated EPSPs (73%) or IPSPs (27%) as well as coincident EPSPs and IPSPs (11%). Indicating that these IPSPs were monosynaptic, light-evoked EPSPs and IPSPs had the same latency and the IPSPs persisted in the presence of ionotropic glutamate receptor antagonists. Overall, our results indicate that the BLA sends a mixed, glutamatergic-GABAergic projection to MD, which likely influences coordination of activity between BLA, MD, and medial prefrontal cortex. An important challenge for future studies will be to examine the connections formed by MD-projecting glutamatergic and GABAergic BLA cells with each other and other populations of BLA cells.SIGNIFICANCE STATEMENT The mediodorsal thalamic nucleus (MD) and basolateral amygdala (BLA) send convergent projections to the medial prefrontal cortex. Although many studies reported that the BLA also projects to MD, there is conflicting evidence as to whether this projection is glutamatergic or GABAergic. By combining tract tracing, immunohistochemistry, optogenetics, and patch clamp recordings in vitro, we found that BLA neurons projecting to MD not only include glutamatergic cells, but also long-range GABAergic neurons. Differential recruitment of these two contingents of cells likely influences coordination of activity between the BLA, MD, and medial prefrontal cortex.

A brainstem circuit for nausea suppression.

SUMMARYNausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects blood-borne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through γ-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention.

A Foundation for the Neural Control of Viral Fever

Fever, an acute‐phase neurally‐regulated increase in body temperature, is thought to be an adaptive response to optimize host defenses against invading pathogens. However, fever can be an unpleasant symptom of infection and excessive fever (>41oC) can cause irreversible protein denaturation and result in tissue damage, seizure, coma and death. Common therapeutic approaches to fever include the use of non‐steroidal anti‐inflammatory drugs and cyclooxygenase inhibitors, however these approaches are ineffective for reducing prostaglandin‐independent fevers, can interfere with adaptive and innate immune defenses and are contraindicated for use in some viral infections, including SARS‐CoV2. Although research over the past couple of decades has defined the fundamental central nervous system (CNS) pathways necessary for the lipopolysaccharide model of bacterial fever, the CNS circuits mediating viral fevers are almost completely unknown. Double‐stranded RNA is an intermediary in viral replication and serves as a pathogen‐associated molecular pattern that elicits the acute phase responses to viral infection. Systemic administration of polyinosinic:polycytidylic acid (poly I:C), a synthetic double stranded RNA, mimics the acute phase responses to viral infection. Neurons in the rostral raphe pallidus area (rRPa) and the dorsomedial hypothalamus play important roles in the control of the sympathetic outflow to thermoeffectors, therefore we hypothesized that neurons in these areas would be essential for fever elicited by a model of viral fever, intravenous (iv) poly I:C. Female and male Sprague Dawley rats anesthetized with urethane and α‐chloralose (for BAT sympathetic nerve recording, n=3) or isoflurane (for cutaneous vasoconstriction experiments, n=3) received iv poly I:C (1mg/kg for experiments with sympathetic nerve recording, or 500μg/kg for experiments for cutaneous vasoconstriction). Systemically administered poly I:C increased BAT SNA (+601 ± 161% baseline), BAT temperature (TBAT; +3.1 ± 0.8°C), and expired CO2 (+0.9 ± 0.2%). Poly I:C also elicited CVC, as observed by reductions in paw or tail skin temperature (TPAW/TTail) (‐2.6 ± 1.2°C). A nanoinjection of the ionotropic glutamate receptor antagonists, AP5 and CNQX in the rRPa reversed poly I:C‐evoked increases in BAT SNA (‐596 ± 128% baseline), TBAT (‐1.3 ± 0.2°C), and expired CO2 (‐0.5 ± 0.1%). Nanoinjection of AP5 and CNQX into the DMH reversed the polyI:C‐evoked reduction in TPAW/TTail (+2.8 ± 1.1°C). These data suggest that the activity of neurons in the rRPa and the DMH are necessary for poly I:C‐induced activation of BAT thermogenesis and cutaneous vasoconstriction. These data define critical components of the neural circuits for poly I:C‐induced fever, highlight differences between the neural circuitry mediating bacterial fever and viral fever, and provide a foundation for elucidating additional brain mechanisms responsible for viral fever.

Substance P Increases the Excitability of Dorsal Motor Nucleus of the Vagus Nerve via Inhibition of Potassium Channels

Increases in the substance P (SP) concentration in the medial portion of the dorsal motor nucleus of the vagus nerve (mDMV) in the brainstem are closely associated with chemotherapy induced nausea and vomiting (CINV). However, the underlying cellular and molecular mechanisms of action are not well understood. In this study, we investigated the effects of SP on mDMV neurons using whole-cell patch-clamp recordings from rat brainstem slices. Application of different concentrations of SP induced tonic and phasic responses. Submicromolar concentrations of induced an inward shift of the holding current by increasing membrane input resistance. The response was mimicked by acidification of the extracellular solution and inhibited by a neurokinin type 1 receptor antagonist. These responses have equilibrium potentials close to the K+ equilibrium potential. In addition, a TWIK-related acid-sensitive K+ channel 3 (TASK-3) inhibitor, PK-THPP, induced responses similar to those produced by submicromolar SP concentrations. Micromolar concentrations of SP facilitated γ-aminobutyric acid (GABA) release but diminished glutamate release; these changes were blocked by a GABAB receptor antagonist and a neurokinin type 3 receptor antagonist, respectively. In current-clamp recordings, submicromolar SP concentrations increased neuronal excitability by depolarizing membrane potentials. However, neither the increase in SP concentration to the micromolar range nor the addition of GABAA and ionotropic glutamate receptor antagonists affected neuronal excitability. Thus, SP increases the excitability of mDMV neurons by inhibiting K+ conductance.

Circuit Contributions to Sensory-Driven Glutamatergic Drive of Olfactory Bulb Mitral and Tufted Cells During Odorant Inhalation.

In the mammalian olfactory bulb (OB), mitral/tufted (MT) cells respond to odorant inhalation with diverse temporal patterns that are thought to encode odor information. Much of this diversity is already apparent at the level of glutamatergic input to MT cells, which receive direct, monosynaptic excitatory input from olfactory sensory neurons (OSNs) as well as a multisynaptic excitatory drive via glutamatergic interneurons. Both pathways are also subject to modulation by inhibitory circuits in the glomerular layer of the OB. To understand the role of direct OSN input vs. postsynaptic OB circuit mechanisms in shaping diverse dynamics of glutamatergic drive to MT cells, we imaged glutamate signaling onto MT cell dendrites in anesthetized mice while blocking multisynaptic excitatory drive with ionotropic glutamate receptor antagonists and blocking presynaptic modulation of glutamate release from OSNs with GABAB receptor antagonists. GABAB receptor blockade increased the magnitude of inhalation-linked glutamate transients onto MT cell apical dendrites without altering their inhalation-linked dynamics, confirming that presynaptic inhibition impacts the gain of OSN inputs to the OB. Surprisingly, blockade of multisynaptic excitation only modestly impacted glutamatergic input to MT cells, causing a slight reduction in the amplitude of inhalation-linked glutamate transients in response to low odorant concentrations and no change in the dynamics of each transient. The postsynaptic blockade also modestly impacted glutamate dynamics over a slower timescale, mainly by reducing adaptation of the glutamate response across multiple inhalations of odorant. These results suggest that direct glutamatergic input from OSNs provides the bulk of excitatory drive to MT cells, and that diversity in the dynamics of this input may be a primary determinant of the temporal diversity in MT cell responses that underlies odor representations at this stage.

Inhibition of the dorsomedial hypothalamus substantially decreases brown adipose tissue sympathetic discharge induced by activation of the lateral habenula.

The lateral habenula (LHb) is an evolutionarily ancient nucleus that plays an important role in the detection of salient/adverse environmental events. We have previously shown that the LHb is involved in brown adipose tissue (BAT) thermogenesis elicited by stressful situations, and that the medullary raphé, a key lower brainstem sympathetic control centre, mediates BAT thermogenesis elicited by stimulating the LHb. Since there are no direct projections from the LHb to the medullary raphé, it is plausible that the dorsomedial hypothalamus (DMH), a brain region known to be important for thermoregulatory responses to stress, is involved in this thermogenic pathway. In this study we aimed to test this possibility. In anaesthetized Sprague-Dawley rats, we recorded electrical discharges directly from sympathetic fibres that innervate BAT, as well as BAT temperature. Injections of bicuculline (1nmol in 100nl), a neuronal activator by disinhibition, into the LHb increased BAT sympathetic nerve discharge by 4.9±1.4dBμV (n=7, P<0.05) and BAT temperature by 1.0±0.1°C (n=7, P<0.01). Subsequent injections of muscimol (0.25nmol in 100nl), a neuronal inhibitor, into the DMH promptly reduced BAT sympathetic nerve discharge by 4.7±1.3 dBμV (n=7, P<0.05) and BAT temperature by 0.3±0.1°C (n=7, P<0.05). Injections of a mixture of the ionotropic glutamate receptor antagonists, DL-2-Amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dioneis (CNQX) into the DMH, after activation of the LHb, also significantly decreased BAT sympathetic nerve discharge and BAT temperature. These results suggest that, for sympathetically-mediated BAT thermogenesis, the DMH is part of the neural circuitry linking the LHb with the medullary raphé.

Roles of glutamate receptors in a novel in vitro model of early, comorbid cerebrovascular, and Alzheimer's diseases.

Systemic multimorbidity is highly prevalent in the elderly and, remarkably, coexisting neuropathological markers of Alzheimer's (AD) and cerebrovascular (CVD) diseases are found at autopsy in most brains of patients clinically diagnosed as AD. Little is known on neurodegeneration peculiar to comorbidities, especially at early stages when pathogenesis may propagate at subclinical levels. We developed a novel in vitro model of comorbid CVD/AD in organotypic hippocampal cultures, by combining oxygen-glucose deprivation (OGD) and exposure to amyloid-Aβ oligomers (AβOs), both applied at levels subtoxic to neurons when used in isolation. We focused on synaptic proteins and the roles of glutamate receptors, which have been implicated in many basic and clinical approaches to either CVD or AD. Subtoxic insults by OGD and AβOs synergized to reduce levels of synaptophysin (SYP) and PSD-95 without cell death, while effects of antagonists of either metabotropic or ionotropic glutamate receptors were distinct from reports in models of isolated CVD or AD. In particular, modulation of glutamate receptors differentially impacted SYP and PSD-95, and antagonists of a single receptor subtype had distinct effects when either isolated or combined. Our findings highlight the complexity of CVD/AD comorbidity, help understand variable responses to glutamate receptor antagonists in patients diagnosed with AD and may contribute to future development of therapeutics based on investigation of the pattern of progressive comorbidity.

Methyl farnesoate regulatory mechanisms underlying photoperiod‐dependent sex determination in the freshwater crustacean Daphnia magna

Freshwater zooplankton Daphnia magna has been widely used in ecotoxicology studies. During the last 20 years, it has been demonstrated that the topical application of juvenile hormone (JH) or JH analogs to mother daphnids induce male offspring production. Based on this finding, an in vivo screening validation method for chemicals with JH agonistic effect has developed. Although this screening system successfully identified a number of JH-like chemicals, molecular mechanisms underlying the male sex-determining process remain largely unknown. To address this issue, we established a reliable male- or female-producing system using Daphnia pulex WTN6 strain by changing the rearing photoperiod. Taking advantage of this rearing system, we successfully found several factors involving male sex determination such as ionotropic glutamate receptors, protein kinase C and pantothenate. Here, we used two D. magna strains that can also control the production of female or male offspring by photoperiod differences as model species for ecotoxicology studies. We demonstrated that either treatment of antagonist of ionotropic glutamate receptors or inhibitor of protein kinase C strongly suppressed male offspring production even under male-producing conditions. Moreover, we revealed that male sex-determining processes are likely diverged between D. magna and D. pulex based on the current experiment. This study provides a fine experimental method for in vivo screening not only JH agonists but also JH antagonists. Moreover, using daphnids with photoperiod-dependent sex determination manner will hugely contribute to understanding the mode-of-action of JH in daphnids.

Stereoselective Synthesis of New (2S,3R)-3-Carboxyphenyl)pyrrolidine-2-carboxylic Acid Analogues Utilizing a C(sp3)-H Activation Strategy and Structure-Activity Relationship Studies at the Ionotropic Glutamate Receptors.

Competitive antagonists for ionotropic glutamate receptors (iGluRs) are highly valuable tool compounds for studying health and disease states in the central nervous system. However, only few subtype selective tool compounds are available and the discovery of antagonists with novel iGluR subtype selectivity profiles remains a profound challenge. In this paper, we report an elaborate structure-activity relationship (SAR) study of the parental scaffold 2,3-trans-3-carboxy-3-phenyl-proline by the synthesis of 40 new analogues. Three synthetic strategies were employed with two new strategies of which one being a highly efficient and fully enantioselective strategy based on C(sp3)-H activation methodology. The SAR study led to the conclusion that selectivity for the NMDA receptors was a general trend when adding substituents in the 5'-position. Selective NMDA receptor antagonists were obtained with high potency (IC50 values as low as 200 nM) and 3-34-fold preference for GluN1/GluN2A over GluN1/GluN2B-D NMDA receptors.

Chronic dietary supplementation with kynurenic acid, a neuroactive metabolite of tryptophan, decreased body weight without negative influence on densitometry and mandibular bone biomechanical endurance in young rats.

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan. KYNA naturally occurs in breast milk and its content increases with lactation, indicating the role of neonatal nutrition in general growth with long-term health effects. KYNA is also an antagonist of ionotropic glutamate receptors expressed in bone cells. The aim of this study was to establish the effects of chronic KYNA supplementation on bone homeostasis in young rats, using mandible as a model bone. Female and male newborn Wistar rats were divided into control and KYNA-administered groups until 60 days of age (25x101 mg/L or 25x102 mg/L in drinking water). Hemimandibles were subjected to densitometry, computed tomography analysis and mechanical testing. Rats supplemented with KYNA at both doses showed a decrease in body weight. There were no effects of KYNA administration and mandible histomorphometry. In males, a significant quadratic effect (P < 0.001) was observed in the densitometry of the hemimandible, where BMD increased in the group supplemented with 2.5x101 mg/L of KYNA. Analysis of mechanical tests data showed that when fracture forces were corrected for bone geometry and rats body weight the improvement of bone material properties was observed in male and female rats supplemented with lower dose of KYNA. This study showed that chronic supplementation with KYNA may limit weight gain in the young, without adversely affecting the development of the skeleton.

Central blockade of the AT1 receptor attenuates pressor effects via reduction of glutamate release and downregulation of NMDA/AMPA receptors in the rostral ventrolateral medulla of rats with stress-induced hypertension.

Glutamatergic activity in the rostral ventrolateral medulla (RVLM), which is an important brain area where angiotensin II (Ang II) elicits its pressor effects, contributes to the onset of hypertension. The present study aimed to explore the effect of central Ang II type 1 receptor (AT1R) blockade on glutamatergic actions in the RVLM of stress-induced hypertensive rats (SIHR). The stress-induced hypertension (SIH) model was established by electric foot shocks combined with noises. Normotensive Sprague-Dawley rats (control) and SIHR were intracerebroventricularly infused with the AT1R antagonist candesartan or artificial cerebrospinal fluid for 14 days. Mean arterial pressure (MAP), heart rate (HR), plasma norepinephrine (NE), glutamate, and the expression of N-methyl-D-aspartic acid (NMDA) receptor subunit NR1, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in the RVLM increased in the SIH group. These increases were blunted by candesartan. Bilateral microinjection of the ionotropic glutamate receptor antagonist kynurenic acid, the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate, or the AMPA/kainate receptors antagonist 6-cyano-7-nitroquinoxaline-2,3-dione into the RVLM caused a depressor response in the SIH group, but not in other groups. NR1 and AMPA receptors expressed in the glutamatergic neurons of the RVLM, and glutamate levels, increased in the intermediolateral column of the spinal cord of SIHR. Central Ang II elicits release of glutamate, which binds to the enhanced ionotropic NMDA and AMPA receptors via AT1R, resulting in activation of glutamatergic neurons in the RVLM, increasing sympathetic excitation in SIHR.

TARP mediation of accelerated and more regular locus coeruleus network bursting in neonatal rat brain slices

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARP) increase neuronal excitability. However, it is unknown how TARP affect rhythmic neural network activity. Here we studied TARP effects on local field potential (LFP) bursting, membrane potential and cytosolic Ca2+ (Cai) in locus coeruleus neurons of newborn rat brain slices. LFP bursting was not affected by the unselective competitive ionotropic glutamate receptor antagonist kynurenic acid (2.5 mM). TARP-AMPAR complex activation with 25 μM CNQX accelerated LFP rhythm 2.2-fold and decreased its irregularity score from 63 to 9. Neuronal spiking was correspondingly 2.3-fold accelerated in association with a 2–5 mV depolarization and a modest Cai rise whereas Cai was unchanged in neighboring astrocytes. After blocking rhythmic activities with tetrodotoxin (1 μM), CNQX caused a 5–8 mV depolarization and also the Cai rise persisted. In tetrodotoxin, both responses were abolished by the non-competitive AMPAR antagonist GYKI 53655 (25 μM) which also reversed stimulatory CNQX effects in control solution. The CNQX-evoked Cai rise was blocked by the L-type voltage-activated Ca2+ channel inhibitor nifedipine (100 μM). The findings show that ionotropic glutamate receptor-independent neonatal locus coeruleus network bursting is accelerated and becomes more regular by activating a TARP-AMPAR complex. The associated depolarization-evoked L-type Ca2+ channel-mediated neuronal Cai rise may be pivotal to regulate locus coeruleus activity in cooperation with SK-type K+ channels. In summary, this is the first demonstration of TARP-mediated stimulation of neural network bursting. We hypothesize that TARP-AMPAR stimulation of rhythmic locus coeruleus output serves to fine-tune its control of multiple brain functions thus comprising a target for drug discovery.

Blocking of Dendrodendritic Inhibition Unleashes Widely Spread Lateral Propagation of Odor-evoked Activity in the Mouse Olfactory Bulb

The olfactory circuitry in mice involves a well-characterized, vertical receptor type-specific organization, but the localized inhibitory effect from granule cells on action potentials that propagate laterally in secondary dendrites of mitral cell remains open to debate. To understand the functional dynamics of the lateral (horizontal) circuits, we analyzed odor-induced signaling using transgenic mice expressing a genetically encoded Ca2+ indicator specifically in mitral/tufted and some juxtaglomerular cells. Optical imaging of the dorsal olfactory bulb (dOB) revealed specific patterns of glomerular activation in response to odor presentation or direct electric stimulation of the olfactory nerve (ON). Application of a mixture of ionotropic and metabotropic glutamate receptor antagonists onto the exposed dOB completely abolished the responses to direct stimulation of the ON as well as discrete odor-evoked glomerular responses patterns, while a spatially more widespread response component increased and expanded into previously nonresponsive regions. To test whether the widespread odor response component represented signal propagation along mitral cell secondary dendrites, an NMDA receptor antagonist alone was applied to the dOB and was found to also increase and expand odor-evoked response patterns. Finally, with dOB excitatory synaptic transmission completely blocked, application of 1 mM muscimol (a GABAA receptor agonist) to a circumscribed volume in the deep external plexiform layer (EPL) induced an odor non-responsive area. These results indicate that odor stimulation can activate olfactory reciprocal synapses and control lateral interactions among olfactory glomerular modules along a wide range of mitral cell secondary dendrites by modulating the inhibitory effect from granule cells.

Reflex regulation of breathing by the paratrigeminal nucleus via multiple bulbar circuits.

Sensory neurons of the jugular vagal ganglia innervate the respiratory tract and project to the poorly studied medullary paratrigeminal nucleus. In the present study, we used neuroanatomical tracing, pharmacology and physiology in guinea pig to investigate the paratrigeminal neural circuits mediating jugular ganglia-evoked respiratory reflexes. Retrogradely traced laryngeal jugular ganglia neurons were largely (> 60%) unmyelinated and expressed the neuropeptide substance P and calcitonin gene-related peptide, although a population (~ 30%) of larger diameter myelinated jugular neurons was defined by the expression of vGlut1. Within the brainstem, vagal afferent terminals were confined to the caudal two-thirds of the paratrigeminal nucleus. Electrical stimulation of the laryngeal mucosa evoked a vagally mediated respiratory slowing that was mimicked by laryngeal capsaicin application. These laryngeal reflexes were modestly reduced by neuropeptide receptor antagonist microinjections into the paratrigeminal nucleus, but abolished by ionotropic glutamate receptor antagonists. D,L-Homocysteic acid microinjections into the paratrigeminal nucleus mimicked the laryngeal-evoked respiratory slowing, whereas capsaicin microinjections evoked a persistent tachypnoea that was insensitive to glutamatergic inhibition but abolished by neuropeptide receptor antagonists. Extensive projections from paratrigeminal neurons were anterogradely traced throughout the pontomedullary respiratory column. Dual retrograde tracing from pontine and ventrolateral medullary termination sites, as well as immunohistochemical staining for calbindin and neurokinin 1 receptors, supported the existence of different subpopulations of paratrigeminal neurons. Collectively, these data provide anatomical and functional evidence for at least two types of post-synaptic paratrigeminal neurons involved in respiratory reflexes, highlighting an unrecognised complexity in sensory processing in this region of the brainstem.

Astrocytic rather than neuronal P2X7 receptors modulate the function of the tri-synaptic network in the rodent hippocampus

Whole-cell patch clamp recordings demonstrated that in the dentate gyrus (DG) as well as in the CA3 area of mouse hippocampal slices the prototypic P2X7 receptor (R) agonist dibenzoyl-ATP (Bz-ATP) induced inward current responses both in neurons and astrocytes. Whereas the selective P2X7R antagonist A438079 strongly inhibited both neuronal and astrocytic currents, a combination of ionotropic glutamate receptor (CNQX, AP-5) and GABAA-R (gabazine) antagonists depressed the Bz-ATP-induced current responses in the DG (granule cells) and CA3 neurons only. It was concluded that Bz-ATP activated astrocytic P2X7Rs and thereby released glutamate and GABA to stimulate nearby neurons. The residual A438079-resistant current response of astrocytes was suggested to be due to the stimulation of P2XRs of the non-P2X7-type. Further, we searched for presynaptic P2X7Rs at the axon terminals of DG and CA3 pyramidal neurons innervating CA3 and CA1 cells, respectively. Bz-ATP potentiated the frequency of spontaneous postsynaptic currents (sPSCs) in CA1 but not CA3 pyramidal cells. However, the Bz-ATP effect in CA1 cells was inhibited by gabazine or the astrocytic toxin fluorocitrate suggesting stimulation of P2X7Rs at stratum radiatum astrocytes located near to interneurons and synapsing onto CA1 neurons. Our data suggest that functional P2X7Rs are missing at neurons in the tri-synaptic network of the rodent hippocampus, but are present at nearby astrocytes indirectly regulating network activity.