Circadian rhythm dysfunction in the suprachiasmatic nucleus : Effects of Trypanosoma brucei brucei infection and inflammatory cytokines

Alterations in the mammalian circadian pacemaker, the hypothalamic suprachiasmatic nuclei (SCN), were studied in an experimental rat model of African trypanosomiasis, or sleeping sickness, caused by infections with subspecies of Trypanosoma brucei (T b). Characteristic signs of the disease are marked disturbances in circadian rhythms, such as a fragmentation of the sleep-wake cycle. Circadian rhythms are mastered from neurons in the SCN, which have an endogenous rhythm of spontaneous firing that is increased during the subjective day. The SCN rhythm can be recorded as spontaneous single unit activity in slice preparations containing the SCN. This activity was recorded in slices from controls and rats infected with T. b. brucei. The rhythm in spontaneous neuronal activity was markedly altered in trypanosome-infectec rats, with a lower average frequency and a phase advance of the peak. No structural differences in the retinal afferents were detected The spontaneous postsynaptic activity was analyzed in slices by whole cell patch clamp recordings of S CN neurons. The inhibitory and excitatory postsynaptic events were identified as primarily gamma- ammobutyric acid (GABA) A and alpha-amino-hydroxy-5-mothyhsoxazole-4propionic acid (AMPA) receptor- dependent. The inhibitory and excitatory activity was compared between the subjective day and night in control rats. No significant difference was detected in amplitude or frequency of inhibitory synaptic events, but die frequency of excitatory events was significantly increased during the subjective day. In slices from rats infected with T. b. brucei, the frequency of excitatory events was significantly lower during the subjective day as compared with control rats. In addition, the protein expressions of AMPA glutamate receptor subunit 2 & 3, and N-methyl-D-aspartate receptor channel subunit zeta1 (NMDAR1), were decreased in trypanosome-infected rats. Invasion of trypanosomes causes a substantial release of several cytokines as an immune response to the infection, for instance tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma, which act in synergy. TNF-alpha and IFN-gamma in combination with bacterial lipopolysaccharide were added to SCN slices and the spontaneous firing was examined. The cytokines altered the rhythm in firing frequency; i. c. caused an abolishment or shift of the peak. The transcript and protein of the IFN-gamma receptor (IFN-gammaR) was detected in the SCN. The molecular identity of the receptor transcript was determined, confirming that the receptor molecule was identical to the IFN-gammaR in the immune system. The expression of the receptor, protein showed daily variations with a peak of expression during the early subjective night. The cyclic variation was abolished and the protein levels were increased in rats held in constant darkness. The postnatal development of the IFN- gammaR protein was studied. At postnatal day (P) 1 the protein was distributed throughout the entire nuclei, but relocated to the ventrolateral retinorecipient subdivisions of the SCN between P l 1 and P20. In conclusion, the present findings demonstrate that T. b. brucei dysregulates the endogenous rhythm in SCN activity, which probably alters the circadian output and may be manifested as a fragmentation of the sleep-wake cycle. Further, the SCN contain glutamatergic synapses that display an increase in activity during the subjective day in vitro. This activity is decreased in slices from trypanosome-infected rats, possibly explaining the observed alteration in spontaneous firing. Cytokines released during trypanosome- infections, such as IFN-gamma may affect protein expression of glutamate receptors and glutamatergic postsynaptic transmission via its receptor, which is located in the ventralateral regions of the SCN

Differential Phase Arrangement of Cellular Clocks along the Tonotopic Axis of the Mouse Cochlea Ex Vivo

Topological distributions of individual cellular clocks have not been demonstrated in peripheral organs. The cochlea displays circadian patterns of core clock gene expression [1, 2]. PER2 protein is expressed in the hair cells and spiral ganglion neurons of the cochlea in the spiral ganglion neurons [1]. To investigate the topological organization of cellular oscillators in the cochlea, we recorded circadian rhythms from mouse cochlear explants using highly sensitive real-time tracking of PER2::LUC bioluminescence. Here, we show cell-autonomous and self-sustained oscillations originating from hair cells and spiral ganglion neurons. Multi-phased cellular clocks were arranged along the length of the cochlea with oscillations initiating at the apex (low-frequency region) and traveling toward the base (high-frequency region). Phase differences of 3 hr were found between cellular oscillators in the apical and middle regions and from isolated individual cochlear regions, indicating that cellular networks organize the rhythms along the tonotopic axis. This is the first demonstration of a spatiotemporal arrangement of circadian clocks at the cellular level in a peripheral organ. Cochlear rhythms were disrupted in the presence of either voltage-gated potassium channel blocker (TEA) or extracellular calcium chelator (BAPTA), demonstrating that multiple types of ion channels contribute to the maintenance of coherent rhythms. In contrast, preventing action potentials with tetrodotoxin (TTX) or interfering with cell-to-cell communication the broad-spectrum gap junction blocker (CBX [carbenoxolone]) had no influence on cochlear rhythms. These findings highlight a dynamic regulation and longitudinal distribution of cellular clocks in the cochlea

Binding site feature description of 2-substituted benzothiazoles as potential AcrAB-TolC efflux pump inhibitors in E. coli

The resistance-nodulation-division (RND) family efflux pumps are important in the antibiotic resistance of Gram-negative bacteria. However, although a number of bacterial RND efflux pump inhibitors have been developed, there has been no clinically available RND efflux pump inhibitor to date. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combinations with ciprofloxacin (CIP) against the AcrAB-TolC overexpressor Escherichia coli AG102 clinical strain. The results indicated that the BSN compounds did not show intrinsic antimicrobial activity when tested alone. However, when used in combinations with CIP, a reversal in the antibacterial activity of CIP with up to 10-fold better MIC values was observed. In order to describe the binding site features of these BSN compounds with AcrB, docking studies were performed using the CDocker method. The performed docking poses and the calculated binding energy scores revealed that the tested compounds BSN-006, BSN-023, and BSN-004 showed significant binding interactions with the phenylalanine-rich region in the distal binding site of the AcrB binding monomer. Moreover, the tested compounds BSN-006 and BSN-023 possessed stronger binding energies than CIP, verifying that BSN compounds are acting as the putative substrates of AcrB. © 2015, Taylor & Francis