Distinct Modulation of Spontaneous and GABA-Evoked Gating by Flurazepam Shapes Cross-Talk Between Agonist-Free and Liganded GABAA Receptor Activity

GABAA receptors (GABAARs) play a crucial inhibitory role in the CNS. Benzodiazepines (BDZs) are positive modulators of specific subtypes of GABAARs, but the underlying mechanism remains obscure. Early studies demonstrated the major impact of BDZs on binding and more recent investigations indicated gating, but it is unclear which transitions are affected. Moreover, the upregulation of GABAAR spontaneous activity by BDZs indicates their impact on receptor gating but the underlying mechanisms remain unknown. Herein, we investigated the effect of a BDZ (flurazepam) on the spontaneous and GABA-induced activity for wild-type (WT, α1β2γ2) and mutated (at the orthosteric binding site α1F64) GABAARs. Surprisingly, in spite of the localization at the binding site, these mutations increased the spontaneous activity. Flurazepam (FLU) upregulated this activity for mutants and WT receptors to a similar extent by affecting opening/closing transitions. Spontaneous activity affected GABA-evoked currents and is manifested as an overshoot after agonist removal that depended on the modulation by BDZs. We explain the mechanism of this phenomenon as a cross-desensitization of ligand-activated and spontaneously active receptors. Moreover, due to spontaneous activity, FLU-pretreatment and co-application (agonist + FLU) protocols yielded distinct results. We provide also the first evidence that GABAAR may enter the desensitized state in the absence of GABA in a FLU-dependent manner. Based on our data and model simulations, we propose that FLU affects agonist-induced gating by modifying primarily preactivation and desensitization. We conclude that the mechanisms of modulation of spontaneous and ligand-activated GABAAR activity concerns gating but distinct transitions are affected in spontaneous and agonist-evoked activity

Comparative evaluation of cytokine and acute-phase protein concentrations in sera of dairy cows with subclinical and clinical ketosis as a different view of the causes of the disease

Background and Aim: Ketosis is a common disease occurring during the first stage of lactation in highly productive dairy cows. The aim of the present study was the comparative assessment of selected pro-inflammatory cytokines (including tumor necrosis factor-α [TNF-α] and interleukin 6 [IL-6]), anti-inflammatory cytokines (including IL-10), and acute-phase proteins (APPs) (including haptoglobin [Hp] and serum amyloid A [SAA]), in the sera of cows with subclinical ketosis (SCK), in those with clinical ketosis (CK), and in healthy cows. Materials and Methods: Thirty dairy cows of Holstein-Friesian breed were investigated. The cows were divided into three groups depending on the serum β-hydroxybutyric acid (BHBA) level. The control, SCK, and CK groups included healthy cows, cows with SCK, and cows with CK, respectively. BHBA concentration in blood serum was determined using colorimetric method. The blood serum was used for proper tests. Cytokine (TNF-α, IL-6, and IL-10) and APPs (SAA and Hp) concentrations in the investigated samples were determined by enzyme-linked immunosorbent assay method. Results: The SCK group had significantly higher TNF-α, IL-6; IL-10, and SAA values than had the CK group (p<0.01). The SCK group had a lower Hp concentration than had the CK group (p<0.05). Conclusion: This study showed that the inflammation intensity is higher in the initial phase of the disease and decreases during the advancement, probably due to active anti-inflammatory mechanisms (an increase of IL-10 concentration), which protect animal organism from self-destruction. On the basis of our study, it can be assumed that ketosis development in dairy cows was preceded by the systemic inflammation that may influence the progress of this disease

PIEZO2-dependent rapid pain system in humans and mice.

The PIEZO2 ion channel is critical for transducing light touch into neural signals but is not considered necessary for transducing acute pain in humans. Here, we discovered an exception - a form of mechanical pain evoked by hair pulling. Based on observations in a rare group of individuals with PIEZO2 deficiency syndrome, we demonstrated that hair-pull pain is dependent on PIEZO2 transduction. Studies in control participants showed that hair-pull pain triggered a distinct nocifensive response, including a nociceptive reflex. Observations in rare Aβ deafferented individuals and nerve conduction block studies in control participants revealed that hair-pull pain perception is dependent on Aβ input. Single-unit axonal recordings revealed that a class of cooling-responsive myelinated nociceptors in human skin is selectively tuned to painful hair-pull stimuli. Further, we pharmacologically mapped these nociceptors to a specific transcriptomic class. Finally, using functional imaging in mice, we demonstrated that in a homologous nociceptor, Piezo2 is necessary for high-sensitivity, robust activation by hair-pull stimuli. Together, we have demonstrated that hair-pulling evokes a distinct type of pain with conserved behavioral, neural, and molecular features across humans and mice