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Local glutamatergic transmission in the RTN/pFRG is critical for active expiration and sympathetic overactivity during hypercapnia
Barnett WH, Molkov YI, Lemes E, Falqueto B, Colombari E, Takakura AT, Moreira TS, Zoccal DB (2017) Local glutamatergic transmission in the RTN/pFRG is critical for active expiration and sympathetic overactivity during hypercapnia. Neuroscience 2017 Abstracts 233.1 / FF22. Society for Neuroscience, Washington, DC.
Summary: The retrotrapezoid nucleus (RTN) contains chemosensitive cells that distribute CO -dependent excitatory drive to the brainstem respiratory network. This drive facilitates the function of the respiratory central pattern generator (CPG), modulates sympathetic activity and determines the emergence of active expiration during hypercapnia via activation of the late expiratory (late-E) oscillator in the parafacial respiratory group (pFRG). However, the microcircuitry responsible for distribution of the chemoreflex signal to the pFRG and the respiratory CPG is not well understood. Previously, we developed a computational model of the brainstem respiratory network, which was subsequently extended to include the central and peripheral chemoreflexes as well as presympathetic circuits. We present here experiments performed on the decerebrated, arterially-perfused in situ rat, aimed to test a key assumption of this model that chemosensitive and late-E neurons in the RTN/pFRG are two distinct populations, and the latter receives local glutamatergic input from the former. The model predicts: (1) suppression of RTN chemosensitive neurons will diminish the changes to the respiratory pattern and the emergence of active expiration associated with hypercapnia; (2) the disruption of local glutamatergic neurotransmission in the RTN will specifically suppress active expiration and the appearance of late-E discharges in the sympathetic motor output. To test prediction (1) we lesioned NK1 -positive chemosensitive neurons of the RTN with microinjections of substance P-saporin (SSP-SAP) conjugate. This suppressed the emergence of late-E activity in abdominal (AbN) and sympathetic nerves, and attenuated the increase in phrenic burst amplitude during hypercapnia. However, SSP-SAP and control animals exhibited late-E AbN activity in response to peripheral chemoreflex activation. Prediction (2) was tested with bilateral microinjections of kynurenic acid (Kyn, 100 mM) in the RTN/pFRG, which suppressed the emergence of late-E AbN activity but not the change in phrenic nerve amplitude during hypercapnia. Our results support the notion that RTN chemosensitive neurons are critical for inspiratory and expiratory reflex responses to hypercapnia. Our findings indicate that activation of late-E neurons in the pFRG during hypercapnia requires glutamatergic inputs from a separate neuronal population in the RTN that intrinsically detects changes in CO . During peripheral chemoreflex stimulation, pFRG late-E neurons are activated via excitatory pathways bypassing the RTN central chemoreceptors. We recapitulate these results in our computational model.
Related Products: SSP-SAP (Cat. #IT-11)
Glutamate and adenosine, basal forebrain and cortex: Cross-talk during prolonged wakefulness
Larin AA, Karpova SA, McCarley RW, Basheer R, Kalinchuk AV (2017) Glutamate and adenosine, basal forebrain and cortex: Cross-talk during prolonged wakefulness. Neuroscience 2017 Abstracts 72.2 /KK24. Society for Neuroscience, Washington, DC.
Summary: Recently we described a biochemical cascade which is critical in promoting recovery sleep (RS) after sleep deprivation (SD). It is initially triggered in the basal forebrain (BF) and later in the prefrontal cortex (PFC). This cascade includes production of inducible nitric oxide synthase (iNOS)-dependent NO followed by an increase in adenosine (AD). We hypothesized that iNOS induction is triggered by an increase in extracellular glutamate (Glu), and that the increase in AD prevents further rise in Glu via its inhibitory action on AD A1 receptor (A1R). To test this hypothesis, during 8h of SD, we first examined the time course of Glu and AD in BF/PFC. Further, to investigate the role of BF Glu receptors (GluRs) in this cascade, we measured the changes in BF/PFC AD and NREMs/delta after: a) stimulating BF GluRs by NMDA or AMPA without SD; b) blocking BF GluRs during SD by NMDAR or AMPAR selective antagonists. Finally, we measured Glu in the BF/PFC after blocking A1R. Furthermore, to determine the cellular target of glutamate effects, we examined the effects BF AMPA infusion on BF/PFC AD and NREMs/delta after BF cholinergic (ChBF) lesions using 192 IgG-saporin. Male rats were implanted with EEG/EMG recording electrodes and microdialysis guide cannulae targeting the BF and PFC. Microdialysis samples were collected during 8h SD and/or drug infusion. AD and Glu were measured using high performance liquid chromatography (HPLC) and ultra HPLC. To block NMDAR/AMPAR/A1R we used dizoclipine (MK-801)/6,7- dinitroquinoxaline-2,3-dione (DNQX)/8 cyclopentyltheophylline (CPT), respectively. 1) In the BF, Glu dramatically increased at the beginning of SD, followed by increase in AD at 2 h of SD. When AD maximized at 4 h of SD, Glu concurrently decreased to baseline. High AD levels were maintained till the end of SD. In the PFC, Glu significantly increased within 2h of SD. When AD increased at 5 h of SD, Glu returned to the baseline. 2) BF AMPA mimicked the effects of SD by increasing AD in both BF and PFC. NREMs/delta increased post AMPA-infusion. NMDA was not effective. 3) BF DNQX prevented AD increase during SD in BF/PFC and attenuated RS. MK-801 did not show any effect. 4) CPT Infusion to the BF/PFC induced dramatic increase in Glu till the end of SD. 5) Lesion of ChBF prevented BF/PFC AD increase during AMPA infusion and attenuated NREMs/delta post-infusion. A rapid increase in Glu during SD may be a trigger for the induction of iNOS-NO-AD cascade in both the BF and PFC. AD via A1R exerts a negative feedback on Glu neurotransmission, preventing its further rise and potential toxicity during long-term SD. The effect of Glu on SDinduced changes is primarily mediated via AMPAR, located on ChBF cells.
Related Products: 192-IgG-SAP (Cat. #IT-01)
Combine phage antibody display library selection on patient tissue specimens with laser capture microdissection to identify novel human antibodies targeting clinically relevant tumor antigens
Su Y, Bidlingmaier S, Lee NK, Liu B (2018) Combine phage antibody display library selection on patient tissue specimens with laser capture microdissection to identify novel human antibodies targeting clinically relevant tumor antigens. (eds. Hust M, Lim T). In: Phage Display. Methods in Molecular Biology. 1701:331-347. Humana Press, New York, NY. doi: 10.1007/978-1-4939-7447-4_18
Objective: To develop a technology that allows selection of phage antibody display libraries on tumor cells in situ residing in their natural tissue microenvironment.
Summary: Intracellular delivery of Immunotoxin was determined as follows: Immunotoxin was prepared by mixing biotinylated scFv with Streptavidin-ZAP (Cat. #IT-27) at a molar ratio of 1:1 and incubated on ice for 30 min. 50 μl of serially diluted immunotoxin was added to each well and incubated for 96 h at 37°C in 5% CO2. Cell growth medium were carefully removed from each well.
Usage: 100 μl of diluted CCK-8 was added to each well in the 96-well plates and incubated for 1–4 h at 37°C in 5% CO2. The absorbance was measured at 450 nm using a microtiter plate reader and the EC50 value determined using GraphPad Prism.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Exposure to low environmental concentrations of manganese, lead, and cadmium alters the serotonin system of blue mussels.
Fraser M, Fortier M, Foucher D, Roumier P, Brousseau P, Fournier M, Surette C, Vaillancourt C (2018) Exposure to low environmental concentrations of manganese, lead, and cadmium alters the serotonin system of blue mussels. Environ Toxicol Chem 37:192-200. doi: 10.1002/etc.3942 PMID: 28796292
Objective: To determine the effects of exposure to Mn, Pb, or Cd on serotonin levels, monoamine oxidase (MAO) activity, and serotonin transporter (SERT) levels in the blue mussel Mytilus edulis.
Summary: The results clearly indicate that low environmental concentrations of Mn, Pb, and Cd affect the serotonin system in blue mussels.
Usage: Membrane was exposed to anti-SERT primary antibody diluted 1:1000 in 5% PBS skimmed milk for 1.5 h at room temperature, then rinsed twice in deionized water, washed 3 times for 5 min in PBS, and rinsed 4 times in deionized water.
Related Products: SERT Mouse Monoclonal (Cat. #AB-N40)
Angiotensin II induces monocyte chemoattractant protein-1 expression by increasing reactive oxygen species-mediated activation of the nuclear factor‑κB signaling pathway in osteoblasts
Wang C, Zhang C, Zhou F, Gao L, Wang Y, Wang C, Zhang Y (2018) Angiotensin II induces monocyte chemoattractant protein-1 expression by increasing reactive oxygen species-mediated activation of the nuclear factor‑κB signaling pathway in osteoblasts. Mol Med Rep 17:1166-1172. doi: 10.3892/mmr.2017.7971 PMID: 29115506
Objective: To investigate the effect of angiotensin II (Ang II) on monocyte chemoattractant protein‑1 (MCP‑1) expression and the underlying mechanism in osteoblasts.
Summary: These data indicate that Ang II-enhanced ROS production and activated NF‑κB signaling via AT1R, thus upregulating MCP‑1 expression in osteoblasts.
Usage: Western Blot; the membranes were blocked with 5% fat‑free milk and probed with anti‑AT1R (1:600).
Related Products: Angiotensin II receptor (AT-1R) Rabbit Polyclonal, affinity-purified (Cat. #AB-N27AP)
Neutrophils are critical for myelin removal in a peripheral nerve injury model of Wallerian degeneration
Lindborg JA, Mack M, Zigmond RE (2017) Neutrophils are critical for myelin removal in a peripheral nerve injury model of Wallerian degeneration. J Neurosci 37(43):10258-10277. doi: 10.1523/JNEUROSCI.2085-17.2017 PMID: 28912156
Objective: To characterize Wallerian degeneration (WD) in male Ccr2−/−mice and identify a compensatory mechanism of WD that is facilitated primarily by neutrophils.
Summary: The data have shown that neutrophils play an impressive role in myelin removal during WD. Neutrophil-specific depletion severely abates nerve debris clearance not only in Ccr2−/− mice, but in WT mice as well. The findings show a novel role for neutrophils that better elucidates the process of WD in the peripheral nervous system.
Usage: immunohistochemistry (1:400)
Related Products: NGFR (mu p75) Rabbit Polyclonal, affinity-purified (Cat. #AB-N01AP)
Increase in cortical endocannabinoid signaling in a rat model of basal forebrain cholinergic dysfunction
Llorente-ovejero A, Manuel I, Giralt MT, Rodríguez-puertas R (2017) Increase in cortical endocannabinoid signaling in a rat model of basal forebrain cholinergic dysfunction. Neuroscience 362:206-218.. doi: 10.1016/j.neuroscience.2017.08.008
Objective: To evaluate the eCB signaling in relation to the memory impairment induced in adult rats following a specific cholinergic lesion of the basal forebrain.
Summary: CB1 receptors present in presynaptic GABAergic terminals in the hippocampus are down regulated, but not those in cortical glutamatergic synapses.
Usage: 192-IgG-SAP was dissolved in aCSF under aseptic conditions to a final concentration of 130 ng/ml. aCSF or 192-IgG-SAP was bilaterally injected (1 ml/hemisphere) at a constant rate of 0.2 ml/min.
Related Products: 192-IgG-SAP (Cat. #IT-01)
Site-specific regulation of P2X7 receptor function in microglia gates morphine analgesic tolerance.
Leduc-Pessah H, Weilinger N, Fan C, Burma N, Thompson R, Trang T (2017) Site-specific regulation of P2X7 receptor function in microglia gates morphine analgesic tolerance. J Neurosci 37:10154-10172.. doi: 10.1523/JNEUROSCI.0852-17.2017
Summary: By selectively ablating microglia in the spinal cord using a Mac-1-SAP the authors demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats.
Usage: Mac-1-SAP or unconjugated Saporin control (15 μg) was administered by intrathecal injection.
Related Products: Mac-1-SAP mouse/human (Cat. #IT-06), Saporin (Cat. #PR-01)
Selective cholinergic depletion of pedunculopontine tegmental nucleus aggravates freezing of gait in parkinsonian rats
Xiao H, Li M, Cai J, Li N, Zhou M, Wen P, Xie Z, Wang Q, Chang J, Zhang W (2017) Selective cholinergic depletion of pedunculopontine tegmental nucleus aggravates freezing of gait in parkinsonian rats. Neurosci Lett 659:92-98.. doi: 10.1016/j.neulet.2017.08.016
Summary: Many patients with advanced Parkinson’s disease suffer from gait and postural impairments. The authors used Anti-ChAT-SAP (Cat. #IT-42) to specifically lesion neurons in the Pedunculopontine Tegmental Nucleus (PPTg) to examine the impact on gait performance. Adult male rats received either unilateral PPTg cholinergic lesion or bilateral PPTg lesion, at a dose of 250 ng. The authors conclude that the cholinergic neurons of pedunculopontine tegmental nucleus play a vital role in the occurrence of gait freezing in Parkinson’s disease.
Related Products: Anti-ChAT-SAP (Cat. #IT-42)
Sleep-inducing effect of substance P-cholera toxin A subunit in mice.
Zielinski M, Gerashchenko D (2017) Sleep-inducing effect of substance P-cholera toxin A subunit in mice. Neurosci Lett 659:44-47.. doi: 10.1016/j.neulet.2017.08.066
Objective: To determine the effects of selectively activating SP-expressing brain cells on sleep regulation in mice.
Summary: ICV administration of SP-CTA produces increased amounts of NREM sleep but induces sleep fragmentation.
Usage: 1 mcg/mcl icv.
Related Products: SP-CTA (Cat. #IT-39)