scholarly journals Contribution of GlyR α3 Subunits to the Sensitivity and Effect of Ethanol in the Nucleus Accumbens

2021 ◽  
Vol 14 ◽  
Author(s):  
Loreto S. San Martin ◽  
Lorena Armijo-Weingart ◽  
Anibal Araya ◽  
Gonzalo E. Yévenes ◽  
Robert J. Harvey ◽  
...  
Keyword(s):  
Nucleus Accumbens ◽  
Glycine Receptor ◽  
Drinking Behavior ◽  
Hek293 Cells ◽  
Knock Out ◽  
Reward Circuitry ◽  
Loss Of Righting Reflex ◽  
Electrophysiological Recordings ◽  
Righting Reflex ◽  
Current Densities

The glycine receptor (GlyR), a ligand-gated ion channel, is critical for inhibitory neurotransmission in brainstem, spinal cord, and in supraspinal regions. Recent data from several laboratories have shown that GlyRs are expressed in the brain reward circuitry and that α1 and α2 are the principal subunits expressed in the nucleus accumbens (nAc). In the present study, we studied the sensitivity to ethanol of homomeric and heteromeric α3 GlyR subunits in HEK293 cells and dissociated neurons from the nAc. Finally, we explored ethanol-related behaviors in a Glra3 knockout mouse (Glra3–/–). Studies in HEK293 cells showed that while homomeric α3 GlyR subunits were insensitive to ethanol, heteromeric α3β GlyR subunits showed higher sensitivity to ethanol. Additionally, using electrophysiological recordings in dissociated accumbal neurons, we found that the glycine current density increased in Glra3–/– mice and the GlyRs were less affected by ethanol and picrotoxin. We also examined the effect of ethanol on sedation and drinking behavior in Glra3–/– mice and found that the duration in the loss of righting reflex (LORR) was unchanged compared to wild-type (WT) mice. On the other hand, using the drinking in the dark (DID) paradigm, we found that Glra3–/– mice have a larger ethanol consumption compared to WT mice, and that this was already high during the first days of exposure to ethanol. Our results support the conclusion that heteromeric α3β, but not homomeric α3, GlyRs are potentiated by ethanol. Also, the increase in GlyR and GABAAR mediated current densities in accumbal neurons in the KO mice support the presence of compensatory changes to α3 knock out. The increase in ethanol drinking in the Glra3–/– mice might be associated to the reduction in β and compensatory changes in other subunits in the receptor arrangement.

scholarly journals Dopaminergic Projections From the Ventral Tegmental Area to the Nucleus Accumbens Modulate Sevoflurane Anesthesia in Mice

2021 ◽  
Vol 15 ◽  
Author(s):  
Huan Gui ◽  
Chengxi Liu ◽  
Haifeng He ◽  
Jie Zhang ◽  
Hong Chen ◽  
...  
Keyword(s):  
Nucleus Accumbens ◽  
Ventral Tegmental Area ◽  
Critical Role ◽  
Sevoflurane Anesthesia ◽  
Loss Of Righting Reflex ◽  
Delta Band ◽  
Dopaminergic Pathway ◽  
Righting Reflex ◽  
Ventral Tegmental

The role of the dopaminergic pathway in general anesthesia and its potential mechanisms are still unknown. In this study, we usedc-Fos staining combined with calcium fiber photometry recording to explore the activity of ventral tegmental area (VTA) dopaminergic neurons(VTA-DA) and nucleus accumbens (NAc) neurons during sevoflurane anesthesia. A genetically encoded dopamine (DA) sensor was used to investigate thefunction of the NAc in sevoflurane anesthesia. Chemogenetics and optogenetics were used to explore the role of the VTA-DA in sevofluraneanesthesia. Electroencephalogram (EEG) spectra, time of loss of righting reflex (LORR) and recovery of righting reflex (RORR) were recorded asassessment indicators. We found that VTA-DA and NAc neurons were inhibited during the induction period and were activated during the recoveryperiod of sevoflurane anesthesia. The fluorescence signals of dopamine decreased in the induction of and increased in the emergence from sevoflurane anesthesia.Activation of VTA-DA and the VTADA-NAc pathway delayed the induction and facilitated the emergence accompanying with thereduction of delta band and the augmentation of the gamma band. These data demonstrate that VTA-DA neurons play a critical role in modulating sevofluraneanesthesia via the VTADA-NAc pathway.

Investigation of Potassium Chloride for Euthanasia of Anesthetized Marine Toads (Rhinella marina)

2021 ◽  
Author(s):  
Meghan M. Louis ◽  
Gregory Scott ◽  
Dustin Smith ◽  
Brigid V. Troan ◽  
Larry J. Minter ◽  
...  
Keyword(s):  
Potassium Chloride ◽  
Potassium Concentration ◽  
Plasma Potassium ◽  
Post Treatment ◽  
Spontaneous Movement ◽  
Limits Of Detection ◽  
Rhinella Marina ◽  
Loss Of Righting Reflex ◽  
Righting Reflex ◽  
Technique Failure

Euthanasia techniques in amphibians are poorly described and sparsely validated. This study investigated potassium chloride (KCl) for euthanasia of anesthetized marine toads ( Rhinella marina ). Twenty three toads were immersed in buffered MS-222 (2 g/L) for five minutes (min) beyond loss of righting reflex, manually removed, and randomly administered KCl (n = 6/group) via one of three routes: intracardiac at 10 mEq/kg (IC), intracoelomic at 100 mEq/kg (ICe), or immersion at 4500 mEq/L (IMS) or no treatment (C) (n = 5/group). Doppler sounds were assessed continuously from prior to treatment until two min post-treatment and every five min thereafter until sound cessation or resumption of spontaneous movement. Plasma potassium concentration (K+) was measured at the time of Doppler sound cessation in ICe and IMS. In IC, ICe, IMS, and C, Doppler sound cessation occurred in 4/6, 6/6, 6/6, and 1/5 toads with median (range) or mean + SD times of 0.23 (0-4.65), 17.5 + 9.0, 40.6 + 10.9, and >420 min, respectively. Nonsuccess in 2/6 toads in IC was suspected due to technique failure. Plasma K+ exceeded the limits of detection (>9 mmol/L) in 12/12 toads in ICe and IMS. Five of six toads in C resumed spontaneous movement at median (range) times of 327 (300-367) min. KCl delivered via an intracardiac, intracoelomic, or immersion routes resulted in Doppler sound cessation in 16 of 18 toads and may be appropriate for euthanasia of anesthetized marine toads.

Stereoselective Loss of Righting Reflex in Rats by Isoflurane

2000 ◽  
Vol 93 (3) ◽  
pp. 837-843 ◽  
Author(s):  
Robert Dickinson ◽  
Ian White ◽  
William R. Lieb ◽  
Nicholas P. Franks
Keyword(s):  
Molecular Targets ◽  
Maximal Effect ◽  
General Anesthetics ◽  
General Anesthetic ◽  
Response Curves ◽  
Loss Of Righting Reflex ◽  
Large Numbers ◽  
Righting Reflex ◽  
Volatile Agents

Background Although it is accepted widely that optically active intravenous general anesthetics produce stereoselective effects in animals, the situation regarding volatile agents is confused. Conventional studies with scarce isoflurane enantiomers have been limited to small numbers of animals and produced conflicting results. By injecting these volatile enantiomers intravenously, however, it is possible to study large numbers of animals and obtain reliable results that can help to identify the molecular targets for isoflurane. Methods Pure isoflurane enantiomers were administered intravenously to rats after solubilization in a lipid emulsion. The ability of each enantiomer to produce a loss of righting reflex was determined as a function of dose, and quantal dose-response curves were constructed. In addition, sleep times were recorded with each enantiomer. Chiral gas chromatography was used to measure relative enantiomer concentrations in the brains of rats injected with racemic isoflurane. Results The S(+)-enantiomer was 40 +/- 8% more potent than the R(-)-enantiomer at producing a loss of righting reflex. The S(+)-enantiomer induced longer sleep times (by about 50%) than did the R(-)-enantiomer. Rats anesthetized by a dose of racemic isoflurane sufficient to achieve a half-maximal effect had essentially identical brain concentrations of the two enantiomers. Conclusions The S(+)-enantiomer of the general anesthetic isoflurane is significantly (P < 0.001) more potent than the R(-)-enantiomer at causing a loss of righting reflex in rats. This confirms the view that isoflurane acts by binding to chiral sites. The observed degree of stereoselectivity provides a useful guide for ascertaining from in vitro experiments which molecular targets are most likely to play major roles in the loss of righting reflex caused by isoflurane.

Sleep Deprivation Potentiates the Onset and Duration of Loss of Righting Reflex Induced by Propofol and Isoflurane

2002 ◽  
Vol 97 (4) ◽  
pp. 906-911 ◽  
Author(s):  
Avery Tung ◽  
Martin J. Szafran ◽  
Bryan Bluhm ◽  
Wallace B. Mendelson
Keyword(s):  
Sleep Deprivation ◽  
Neuronal Networks ◽  
Fixed Dose ◽  
Patient Response ◽  
Disk Rotation ◽  
Anesthetic Agents ◽  
Loss Of Righting Reflex ◽  
Separate Control ◽  
Righting Reflex ◽  
Time Required

Background Sleep and anesthesia differ physiologically but produce a similar loss of responsiveness to environmental stimuli. Recent data suggest that neuronal networks active in naturally occurring sleep also play a role in the anesthetized state. Changes in the propensity to sleep may then modify the response to anesthetic agents. The authors tested the hypothesis that sleep-deprived rats would require less anesthetic than rested rats to achieve a similar loss of responsiveness. Methods Rats were subjected to a 24-h period of either sleep deprivation or ad libitum activity. Sleep deprivation was produced by placing rats on a disk that rotated when sleep was detected by electroencephalographic and electromyographic (EEG, EMG) monitoring. A fixed dose of anesthetic agent was then administered, and the time required to induce loss of righting reflex was measured. Anesthetic administration was then stopped, and the time to recovery measured. All rats received both treatments separated by 7 days. Results Sleep deprivation reduced the time to loss of righting reflex by 40% for propofol (P < 0.025) and 55% for isoflurane (P < 0.025) and prolonged the time to recovery. In a separate control experiment, exposure to the deprivation environment but with disk rotation modified to allow adequate sleep did not affect the response to anesthetic administration. Conclusions Sleep deprivation significantly potentiated the ability of inhaled and intravenous anesthetic agents to induce a loss of righting reflex. These results support the hypothesis that neuronal networks active in sleep are also involved in the anesthetized state and suggest that sleep deprivation may partly explain the variability in patient response to anesthesia.

Nalmefene reverses carfentanil-induced loss of righting reflex and respiratory depression in rats

2014 ◽  
Vol 738 ◽  
pp. 153-157 ◽  
Author(s):  
Zheng Yong ◽  
Xiang Gao ◽  
Wentao Ma ◽  
Huajing Dong ◽  
Zehui Gong ◽  
...  
Keyword(s):  
Respiratory Depression ◽  
Loss Of Righting Reflex ◽  
Righting Reflex

The monomeric G proteins AGS1 and Rhes selectively influence Gαi-dependent signaling to modulate N-type (CaV2.2) calcium channels

2008 ◽  
Vol 295 (5) ◽  
pp. C1417-C1426 ◽  
Author(s):  
Ashish Thapliyal ◽  
Roger A. Bannister ◽  
Christopher Hanks ◽  
Brett A. Adams
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Hek293 Cells ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Physiological Conditions ◽  
Channel Inhibition ◽  
Voltage Dependent ◽  
Current Densities

Activator of G protein Signaling 1 (AGS1) and Ras homologue enriched in striatum (Rhes) define a new group of Ras-like monomeric G proteins whose signaling properties and physiological roles are just beginning to be understood. Previous results suggest that AGS1 and Rhes exhibit distinct preferences for heterotrimeric G proteins, with AGS1 selectively influencing Gαi and Rhes selectively influencing Gαs. Here, we demonstrate that AGS1 and Rhes trigger nearly identical modulation of N-type Ca2+ channels (CaV2.2) by selectively altering Gαi-dependent signaling. Whole-cell currents were recorded from HEK293 cells expressing CaV2.2 and Gαi- or Gαs-coupled receptors. AGS1 and Rhes reduced basal current densities and triggered tonic voltage-dependent (VD) inhibition of CaV2.2. Additionally, each protein attenuated agonist-initiated channel inhibition through Gαi-coupled receptors without reducing channel inhibition through a Gαs-coupled receptor. The above effects of AGS1 and Rhes were blocked by pertussis toxin (PTX) or by expression of a Gβγ-sequestering peptide (masGRK3ct). Transfection with HRas, KRas2, Rap1A-G12V, Rap2B, Rheb2, or Gem failed to duplicate the effects of AGS1 and Rhes on CaV2.2. Our data provide the first demonstration that AGS1 and Rhes exhibit similar if not identical signaling properties since both trigger tonic Gβγ signaling and both attenuate receptor-initiated signaling by the Gβγ subunits of PTX-sensitive G proteins. These results are consistent with the possibility that AGS1 and Rhes modulate Ca2+ influx through CaV2.2 channels under more physiological conditions and thereby influence Ca2+-dependent events such as neurosecretion.

Recombinant Human Coagulation Factor IX Expressed in HEK293 Cells: Influence of Serin Phosphorylation and Tyrosine Sulfation on Pharmacokinetic Properties in FIX-Knock-out Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4098-4098
Author(s):  
Ernst Boehm ◽  
Michael Dockal ◽  
Meinhard Hasslacher ◽  
Artur Mitterer ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hek293 Cells ◽  
High Yield ◽  
High Similarity ◽  
Tyrosine Sulfation ◽  
Knock Out ◽  
Pharmacokinetic Properties ◽  
Knock Out Mice ◽  
In Vivo Recovery

Abstract Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

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