Small molecule inhibition of spontaneous calcium oscillations mediated by either NMDA or AMPA will be assessed. Therapeutic protein inhibition of spontaneous calcium oscillations mediated by either NMDA or AMPA will be assessed. Addition of small molecule will inhibit AMPA receptor mediated oscillations. Data presented in FIG. 1 show that, addition of the AMPA receptor antagonist, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3 .mu.M), reduced calcium oscillations by 20% representing the total AMPA response in the assay (FIG. 1AMPA labeled bar shown). Tau antisense oligonucleotide impact on spontaneous calcium oscillations in primary neurons is shown in FIG. 4. The oligomers in neuronal cells showing AMPA-mediated oscillations that are equal to or greater than 75% of the calcium oscillations in the untreated control cells were selected for further analysis. The effect of therapeutic proteins, such as antibodies or antigen-binding fragments thereof, fusion proteins, cytokines, cell surface receptors, hormones or growth factors, on calcium oscillations will be measured using substantially the same method provided in Example 2. To measure primary cortical neuron spontaneous calcium oscillation, rat primary cortical neurons will be prepared.



Intracerebroventricular injections were performed using a Hamilton micro syringe fitted with a 27 or 30-gauge needle, according to the method of Haley and McCormick. Administration Routes-Intra-Cerebroventricular or Intrathecal Injections. The oligomers were administered to mice by either intracerebroventricular (i.c.v.) injection or intrathecal injection. Baseline signal (measurement of intracellular calcium) was read for 100 seconds (1 reading/second) before the addition of anti-sense oligomers. FLIPR signal was read for an additional 200 seconds (1 reading/second) after the addition of oligomer. A second 5 minute post addition plate read (300 one second points) on the FLIPR was conducted to allow for additional data capture. I believe eroticism is one of Gods greatest gifts to humankind, prudishness a bizarre aberration, and not so long ago, hyperbolic warnings about the perils of pornography, whether from my Evangelical Christian or watch live sexcam progressive feminist friends, had me rolling my eyes. My safety has always been a number one priority for them,' she told The Sun, while revealing that she broke the news to them via FaceTime over the weekend. Calculations were performed by measuring the average FLIPR signal over the 300 second read for control (non-treated) wells. Antisense oligomer inhibition of spontaneous calcium oscillations mediated by either NMDA or AMPA was assessed in the presence or absence of 1 mM MgCl.sub.2 (representing 100% control in each case; FIG. 2). Addition of 25 .mu.M antisense oligomers (ASO) inhibited AMPA receptor but not NMDA receptor mediated oscillations (FIG. 2). ASOs, and other oligomers that behaved similarly, were shown to negatively impact central nervous system (CNS) network activity in vivo and electrophysiologic spontaneous neuronal activity in vitro (data not shown).



Calcium oscillations were reduced further, by about 80%, when (NMDA) receptor function was blocked by 1 mM MgCl.sub.2 (FIG. 1 NMDA labeled bar shown). Calcium oscillation reduction in the neuronal cells was measured for the oligomers of the invention and compared with that of the control cells (i.e., the calcium oscillations in the neuronal cells that are not treated with the oligomers). A score of 0 was given for each 1 second read that increase less than 50% of average control value. For treated wells, a scoring system was developed where a score of 1 was given for each 1 second read where signal increase greater than 50% of the average control value (calculated above). Raw data from the 5 minute read was exported and, using Excel, spike amplitude and frequency was calculated. Raw data will be exported, and spike amplitude and frequency will be calculated. Small molecules that reduce calcium oscillations to levels below 70% of control will be expected to negatively impact CNS network activity in vivo and electrophysiologic spontaneous neuronal activity in vitro.



The in vivo tolerability of the oligomers was tested to see how the oligomer was tolerated when injected into an animal. Research protocols were approved by the Bristol-Myers Squibb Company Animal Care and Use Committee. Animals were maintained in accordance with the guidelines of the Animal Care and Use Committee of the Bristol-Myers Squibb Company, and the "Guide for Care and Use of Laboratory Animals" published by the National Institutes of Health. All animals had ad libitum access to food and water throughout the studies. Animals for Tau qPCR and behavioral studies were adult, C57B1/6J female mice (20-30 g; Jackson Laboratories, Bar Harbor, Me.) housed 3-4 per cage. In some cases, male and female transgenic mice (30-40 g) expressing a tau transgene derived from a human PAC, H1 haplotype driven by the tau promoter (Polydoro et. J. Neurosci. (2009) 29(34): 10741-9), and in which the native mouse Tau gene was deleted, were used to assess pharmacodynamic endpoints and tissue drug concentrations.