Seasonal plasticity in GABAA signaling is necessary for restoring phase synchrony in the master circadian clock network.

Annual changes in the environment threaten survival, and numerous biological processes in mammals adjust to this challenge via seasonal encoding by the suprachiasmatic nucleus (SCN). To tune behavior according to day length, SCN neurons display unified rhythms with synchronous phasing when days are short, but will divide into two sub-clusters when days are long. The transition between SCN states is critical for maintaining behavioral responses to seasonal change, but the mechanisms regulating this form of neuroplasticity remain unclear. Here we identify that a switch in chloride transport and GABAA signaling is critical for maintaining state plasticity in the SCN network. Further, we reveal that blocking excitatory GABAA signaling locks the SCN into its long day state. Collectively, these data demonstrate that plasticity in GABAA signaling dictates how clock neurons interact to maintain environmental encoding. Further, this work highlights factors that may influence susceptibility to seasonal disorders in humans.

Synthesis of chiral GABAA receptor subtype selective ligands as potential agents to treat schizophrenia as well as depression.

A series of novel imidazobenzodiazepine analogs of the lead chiral ligand SH-053-2'F-S-CH3 (2), an α2/α3/α5 (Bz)GABA (A)ergic receptor subtype selective ligand, which reversed PCP-induced prepulse inhibition (PPI) of acoustic startle, were synthesized. These chiral (S)-CH3 ligands are targeted for the treatment of schizophrenia and depression. These new ligands were designed by modifying the liable ester functionality in 2 to improve the metabolic stability, cytotoxicity, and activity as compared to 2. Based on the data to date, the most promising ligands are the N-cyclopropyl amide GL-I-55 (8c) and the methyl bioisostere GL-I-65 (9a). The in vitro metabolic stability, cytotoxicity and in vivo locomotor effects are described in this report. Based on these results, 8c and 9a are the most promising for further in vivo pharmacology.

Regulation of System xc(-) by Pharmacological Manipulation of Cellular Thiols.

The cystine/glutamate exchanger (system xc (-)) mediates the transport of cystine into the cell in exchange for glutamate. By releasing glutamate, system xc (-) can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and may protect cells against oxidative stress. We tested two different compounds that deplete primary cortical cultures containing both neurons and astrocytes of intracellular GSH, L-buthionine-sulfoximine (L-BSO), and diethyl maleate (DEM). Both compounds caused significant concentration and time dependent decreases in intracellular GSH levels. However; DEM caused an increase in radiolabeled cystine uptake through system xc (-), while unexpectedly BSO caused a decrease in uptake. The compounds caused similar low levels of neurotoxicity, while only BSO caused an increase in oxidative stress. The mechanism of GSH depletion by these two compounds is different, DEM directly conjugates to GSH, while BSO inhibits γ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. As would be expected from these mechanisms of action, DEM caused a decrease in intracellular cysteine, while BSO increased cysteine levels. The results suggest that negative feedback by intracellular cysteine is an important regulator of system xc (-) in this culture system.

Behavioral assessment of acute inhibition of system xc (-) in rats.

RATIONALE: Gaps in our understanding of glutamatergic signaling may be key obstacles in accurately modeling complex CNS diseases. System xc (-) is an example of a poorly understood component of glutamate homeostasis that has the potential to contribute to CNS diseases. OBJECTIVES: This study aims to determine whether system xc (-) contributes to behaviors used to model features of CNS disease states. METHODS: In situ hybridization was used to map mRNA expression of xCT throughout the brain. Microdialysis in the prefrontal cortex was used to sample extracellular glutamate levels; HPLC was used to measure extracellular glutamate and tissue glutathione concentrations. Acute administration of sulfasalazine (8-16 mg/kg, IP) was used to decrease system xc (-) activity. Behavior was measured using attentional set shifting, elevated plus maze, open-field maze, Porsolt swim test, and social interaction paradigm. RESULTS: The expression of xCT mRNA was detected throughout the brain, with high expression in several structures including the basolateral amygdala and prefrontal cortex. Doses of sulfasalazine that produced a reduction in extracellular glutamate levels were identified and subsequently used in the behavioral experiments. Sulfasalazine impaired performance in attentional set shifting and reduced the amount of time spent in an open arm of an elevated plus maze and the center of an open-field maze without altering behavior in a Porsolt swim test, total distance moved in an open-field maze, or social interaction. CONCLUSIONS: The widespread distribution of system xc (-) and involvement in a growing list of behaviors suggests that this form of nonvesicular glutamate release is a key component of excitatory signaling.

Pyrazolopyridine Inhibitors of B-Raf(V600E). Part 1: The Development of Selective, Orally Bioavailable, and Efficacious Inhibitors.

The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-Raf(V600E) was developed. Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent, selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft model driven by B-Raf(V600E) with no effect on body weight. On the basis of their in vivo efficacy and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.