Propofol modulates phasic and tonic GABAergic currents in spinal ventral horn interneurones.

BACKGROUND: Surgical interventions like skin incisions trigger withdrawal reflexes which require motor neurones and local circuit interneurones in the spinal ventral horn. This region plays a key role in mediating immobilizing properties of the GABAergic anaesthetic propofol. However, it is unclear how propofol modulates GABA(A) receptors in the spinal ventral horn and whether tonic or phasic inhibition is involved. METHODS: Organotypic spinal cord tissue slices were prepared from mice. Whole-cell recordings were performed for quantifying effects of propofol on GABA(A) receptor-mediated phasic transmission and tonic conductance. RESULTS: Propofol increased GABAergic phasic transmission by a prolongation of the decay time constant in a concentration-dependent manner. The amount of the charge transferred per inhibitory post-synaptic current, described by the area under the curve, was significantly augmented by 1 µM propofol (P<0.01). A GABA(A) receptor-mediated tonic current was not induced by 1 µM propofol but at a concentration of 5 µM (P<0.05). CONCLUSIONS: Propofol depresses ventral horn interneurones predominantly by phasic rather than by tonic GABA(A) receptor-mediated inhibition. However, the present results suggest that the involvement of a tonic inhibition might contribute to the efficacy of propofol to depress nociceptive reflexes at high concentrations of the anaesthetic.

Ulcerative colitis and adenocarcinoma of the colon in G alpha i2-deficient mice.

G proteins are involved in cellular signalling and regulate a variety of biological processes including differentiation and development. We have generated mice deficient for the G protein subunit alpha i2 (G alpha i2) by homologous recombination in embryonic stem cells. G alpha i2-deficient mice display growth retardation and develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. Prior to clinical symptoms, the mice show profound alterations in thymocyte maturation and function. The study of these animals should provide important insights into the pathogenesis of ulcerative colitis as well as carcinogenesis.

Effect of maximum exercise on left ventricular deformation and its correlation with cardiopulmonary exercise capacity in competitive athletes.

BACKGROUND: Global longitudinal strain (GLS) and global myocardial work index (GWI) allow early detection of subclinical changes in left ventricular (LV) systolic function. The aim of the study was to investigate the immediate effects of maximum physical exercise by different exercise testing methods on early post exercise LV deformation parameters in competitive athletes and to analyze their correlation with cardiopulmonary exercise capacity. METHODS: To reach maximum physical exercise, cardiopulmonary exercise testing (CPET) was performed by semi-recumbent ergometer in competitive handball players (n = 13) and by treadmill testing in competitive football players (n = 19). Maximum oxygen uptake (VO2max) indexed to body weight (relative VO2max) was measured in all athletes. Transthoracic echocardiography and blood pressure measurements were performed at rest and 5 min after CPET in all athletes. GLS, GWI and their changes before and after CPET (ΔGLS, ΔGWI) were correlated with (relative) VO2max. RESULTS: In handball and football players, GLS and GWI did not differ significantly before and after CPET. There were no significant correlations between GLS and relative VO2max, but moderate correlations were found between ΔGWI and relative VO2max in handball (r = 0.631; P = 0.021) and football players (r = 0.592; P = 0.008). Furthermore, handball (46.7 ml/min*kg ± 4.7 ml/min*kg vs. 37.4 ml/min*kg ± 4.2; P = 0.004) and football players (58.3 ml/min*kg ± 3.7 ml/min*kg vs. 49.7 ml/min*kg ± 6.8; P = 0.002) with an increased ΔGWI after CPET showed a significant higher relative VO2max. CONCLUSION: Maximum physical exercise has an immediate effect on LV deformation, irrespective of the used testing method. The correlation of relative VO2max with ΔGWI in the early post exercise period, identifies ΔGWI as an echocardiographic parameter for characterizing the current individual training status of athletes.

SARS-CoV2 infection: functional and morphological cardiopulmonary changes in elite handball players.

There is increasing evidence of cardiac involvement post-SARS-CoV-2 infections in symptomatic as well as in oligo- and asymptomatic athletes. This study aimed to characterize the possible early effects of SARS-CoV-2 infections on myocardial morphology and cardiopulmonary function in athletes. Eight male elite handball players (27 ± 3.5 y) with past SARS-CoV-2 infection were compared with four uninfected teammates (22 ± 2.6 y). Infected athletes were examined 19 ± 7 days after the first positive PCR test. Echocardiographic assessment of the global longitudinal strain under resting conditions was not significantly changed (- 17.7% vs. - 18.1%). However, magnetic resonance imaging showed minor signs of acute inflammation/oedema in all infected athletes (T2-mapping: + 4.1 ms, p = 0.034) without reaching the Lake-Louis criteria. Spiroergometric analysis showed a significant reduction in VO2max (- 292 ml/min, - 7.0%), oxygen pulse (- 2.4 ml/beat, - 10.4%), and respiratory minute volume (VE) (- 18.9 l/min, - 13.8%) in athletes with a history of SARS-CoV2 infection (p < 0.05, respectively). The parameters were unchanged in the uninfected teammates. SARS-CoV2 infection caused impairment of cardiopulmonary performance during physical effort in elite athletes. It seems reasonable to screen athletes after SARS-CoV2 infection with spiroergometry to identify performance limitations and to guide the return to competition.

Effects of surgical and FFP2/N95 face masks on cardiopulmonary exercise capacity.

BACKGROUND: Due to the SARS-CoV2 pandemic, medical face masks are widely recommended for a large number of individuals and long durations. The effect of wearing a surgical and a FFP2/N95 face mask on cardiopulmonary exercise capacity has not been systematically reported. METHODS: This prospective cross-over study quantitated the effects of wearing no mask (nm), a surgical mask (sm) and a FFP2/N95 mask (ffpm) in 12 healthy males (age 38.1 ± 6.2 years, BMI 24.5 ± 2.0 kg/m2). The 36 tests were performed in randomized order. The cardiopulmonary and metabolic responses were monitored by ergo-spirometry and impedance cardiography. Ten domains of comfort/discomfort of wearing a mask were assessed by questionnaire. RESULTS: The pulmonary function parameters were significantly lower with mask (forced expiratory volume: 5.6 ± 1.0 vs 5.3 ± 0.8 vs 6.1 ± 1.0 l/s with sm, ffpm and nm, respectively; p = 0.001; peak expiratory flow: 8.7 ± 1.4 vs 7.5 ± 1.1 vs 9.7 ± 1.6 l/s; p < 0.001). The maximum power was 269 ± 45, 263 ± 42 and 277 ± 46 W with sm, ffpm and nm, respectively; p = 0.002; the ventilation was significantly reduced with both face masks (131 ± 28 vs 114 ± 23 vs 99 ± 19 l/m; p < 0.001). Peak blood lactate response was reduced with mask. Cardiac output was similar with and without mask. Participants reported consistent and marked discomfort wearing the masks, especially ffpm. CONCLUSION: Ventilation, cardiopulmonary exercise capacity and comfort are reduced by surgical masks and highly impaired by FFP2/N95 face masks in healthy individuals. These data are important for recommendations on wearing face masks at work or during physical exercise.

Molecular and neuronal substrate for the selective attenuation of anxiety.

Benzodiazepine tranquilizers are used in the treatment of anxiety disorders. To identify the molecular and neuronal target mediating the anxiolytic action of benzodiazepines, we generated and analyzed two mouse lines in which the alpha2 or alpha3 GABAA (gamma-aminobutyric acid type A) receptors, respectively, were rendered insensitive to diazepam by a knock-in point mutation. The anxiolytic action of diazepam was absent in mice with the alpha2(H101R) point mutation but present in mice with the alpha3(H126R) point mutation. These findings indicate that the anxiolytic effect of benzodiazepine drugs is mediated by alpha2 GABAA receptors, which are largely expressed in the limbic system, but not by alpha3 GABAA receptors, which predominate in the reticular activating system.

Cortical glutamatergic neurons mediate the motor sedative action of diazepam.

The neuronal circuits mediating the sedative action of diazepam are unknown. Although the motor-depressant action of diazepam is suppressed in alpha1(H101R) homozygous knockin mice expressing diazepam-insensitive alpha1-GABA(A) receptors, global alpha1-knockout mice show greater motor sedation with diazepam. To clarify this paradox, attributed to compensatory up-regulation of the alpha2 and alpha3 subunits, and to further identify the neuronal circuits supporting diazepam-induced sedation, we generated Emx1-cre-recombinase-mediated conditional mutant mice, selectively lacking the alpha1 subunit (forebrain-specific alpha1(-/-)) or expressing either a single wild-type (H) or a single point-mutated (R) alpha1 allele (forebrain-specific alpha1(-/H) and alpha1(-/R) mice, respectively) in forebrain glutamatergic neurons. In the rest of the brain, alpha1(-/R) mutants are heterozygous alpha1(H101R) mice. Forebrain-specific alpha1(-/-) mice showed enhanced diazepam-induced motor depression and increased expression of the alpha2 and alpha3 subunits in the neocortex and hippocampus, in comparison with their pseudo-wild-type littermates. Forebrain-specific alpha1(-/R) mice were less sensitive than alpha1(-/H) mice to the motor-depressing action of diazepam, but each of these conditional mutants had a similar behavioral response as their corresponding control littermates. Unexpectedly, expression of the alpha1 subunit was reduced in forebrain, notably in alpha1(-/R) mice, and the alpha3 subunit was up-regulated in neocortex, indicating that proper alpha1 subunit expression requires both alleles. In conclusion, conditional manipulation of GABA(A) receptor alpha1 subunit expression can induce compensatory changes in the affected areas. Specifically, alterations in GABA(A) receptor expression restricted to forebrain glutamatergic neurons reproduce the behavioral effects seen after a global alteration, thereby implicating these neurons in the motor-sedative effect of diazepam.

Normal sleep homeostasis and lack of epilepsy phenotype in GABA A receptor alpha3 subunit-knockout mice.

Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo-cortical function in physiological and pathophysiological situations.

Hippocampal alpha 5 subunit-containing GABA A receptors are involved in the development of the latent inhibition effect.

Hippocampal GABA(A) receptors containing the alpha 5 subunit have been implicated in the modulation of hippocampal-dependent learning, presumably via their tonic inhibitory influence on hippocampal glutamatergic activity. Here, we examined the expression of latent inhibition (LI)--a form of selective learning that is sensitive to a number of manipulations targeted at the hippocampal formation, in alpha 5(H105R) mutant mice with reduced levels of hippocampal alpha 5-containing GABA(A) receptors. A single pre-exposure to the taste conditioned stimulus (CS) prior to the pairing of the same CS with LiCl-induced nausea was effective in reducing the conditioned aversion against the taste CS in wild-type mice--thus constituting the LI effect. LI was however distinctly absent in male alpha 5(H105R) mutant mice. Hence, a partial loss of hippocampal alpha 5 GABA(A) receptors is sufficient to alter one major form of selective learning, albeit this was not seen in the female. This observed phenotype suggests that specific activation of these extrasynaptic GABA(A) receptors may confer therapeutic potential against the failure to show selectivity in learning by human psychotic patients.

Inhibitory ligand-gated ion channels as substrates for general anesthetic actions.

General anesthetics have been in clinical use for more than 160 years. Nevertheless, their mechanism of action is still only poorly understood. In this review, we describe studies suggesting that inhibitory ligand-gated ion channels are potential targets for general anesthetics in vitro and describe how the involvement of y-aminobutyric acid (GABA)(A) receptor subtypes in anesthetic actions could be demonstrated by genetic studies in vivo.

[The GABA(A) receptor family: possibilities for the development of better anesthetics]. / Die GABA(A)-Rezeptor-Familie: Möglichkeiten für die Entwicklung besserer Anästhetika.

Clinically used anesthetics show amnestic, sedative, hypnotic and immobilizing properties. On a molecular level these drugs affect several receptors in the cell membrane of neurons. By using genetically engineered mice a linkage can now be made between actions on certain receptors and clinically desired and undesired effects. Experiments show that a certain GABA(A) receptor subtype mediates hypnosis and immobility, whereas another subtype is involved in side-effects like sedation and hypothermia. These findings form the basis for the development of new drugs, acting highly specific and with fewer side-effects.

GABA(A) receptor subtypes: dissecting their pharmacological functions.

The enhancement of GABA-mediated synaptic transmission underlies the pharmacotherapy of various neurological and psychiatric disorders. GABA(A) receptors are pluripotent drug targets that display an extraordinary structural heterogeneity: they are assembled from a repertoire of at least 18 subunits (alpha1-6, beta1-3, gamma1-3, delta, epsilon, theta, rho1-3). However, differentiating defined GABA(A) receptor subtypes on the basis of function has had to await recent progress in the genetic dissection of receptor subtypes in vivo. Evidence that the various actions of allosteric modulators of GABA(A) receptors, in particular the benzodiazepines, can be attributed to specific GABA(A) receptor subtypes will be discussed. Such discoveries could open up new avenues for drug development.

GABA(A)-receptor subtypes: a new pharmacology.

The GABA(A) receptor is a pluripotent drug target mediating anxiolytic, sedative, anticonvulsant, muscle relaxant and amnesic activity. These drug actions have now been attributed to defined receptor subtypes. Thus, precise guidelines are available for the development of novel drugs with more selective action and less side effects than those currently in clinical use.

Pathophysiology and pharmacology of GABA(A) receptors.

By controlling spike timing and sculpting neuronal rhythms, inhibitory interneurons play a key role in brain function. GABAergic interneurons are highly diverse. The respective GABA(A) receptor subtypes, therefore, provide new opportunities not only for understanding GABA-dependent pathophysiologies but also for targeting of selective neuronal circuits by drugs. The pharmacological relevance of GABA(A) receptor subtypes is increasingly being recognized. A new central nervous system pharmacology is on the horizon. The development of anxiolytic drugs devoid of sedation and of agents that enhance hippocampus-dependent learning and memory has become a novel and highly selective therapeutic opportunity.

Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABA(A) receptor.

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with ß3 subunits, we tested the hypothesis that etomidate acts through ß3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR ß3-subunit (ß3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by ß3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5ß1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 µM and below), whereas α5ß2γ2L and α5ß3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5ß3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5ß2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons).

The cytosolic G-protein alpha-subunit in human neutrophils responds to treatment with guanine nucleotides and magnesium.

The hydrodynamic properties of the Gi2 alpha-subunit in human neutrophil cytosol were analyzed. The S20,w value was 3.3 S at 30 degrees C and 4.1 S at 4 degrees C. Reconstitution of the cytosolic alpha-subunit with beta gamma-complex purified from porcine brain resulted in an S20,w value of 4.0 S at 30 degrees C and 4.2 S at 4 degrees C. Treatment of cytosol with G-protein-activating agents, GTP gamma S and MgCl2, decreased the S20,w value to 2.4 S at 30 degrees C and 2.9 S at 4 degrees C. Our results indicate that the cytosolic alpha-subunit of neutrophils is an inactive monomeric species at 30 degrees C capable of interacting with G-protein beta gamma-complex and responsive to treatment with activating agents leading to the two activated forms, alpha at 4 degrees C and alpha at 30 degrees C.

Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated alpha-subunits.

Amino acids in the alpha- and gamma-subunits contribute to the benzodiazepine binding site of GABA(A)-receptors. We show that the mutation of a conserved histidine residue in the N-terminal extracellular segment (alpha1H101R, alpha2H101R, alpha3H126R, and alpha5H105R) results not only in diazepam-insensitivity of the respective alphaxbeta2,3gamma2-receptors but also in an increased potentiation of the GABA-induced currents by the partial agonist bretazenil. Furthermore, Ro 15-4513, an inverse agonist at wild-type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABA(A)-receptor subtypes in vivo.

Transgenic and targeted mutant mice in drug discovery.

In the process of drug discovery, different classes of genetically-modified animals are valuable tools for the identification of drug targets, distinction of target isoforms, differentiation of signaling pathways, generation of disease models and toxicological testing. The analysis is refined by methodologies which permit the inducible regulation of gene expression in a time- and tissue-specific manner. Finally, multiplexing techniques and knockout embryonic stem (ES) cell libraries will help to link DNA sequence information to biological function.