All experiments were conducted under protocols approved by the Animal Care and Use Committee of the University of Colorado, Anschutz Medical Campus. Acute horizontal olfactory bulb slices (300C400 m) were prepared following isoflurane anesthesia and decapitation. feedforward excitation is an indirect result of and must cooccur with activation of inhibitory circuitry. Next, to examine the dynamics of the competing signals, we assayed the relationship between the quantity of spikes in eTCs and excitation of MCs or PG cells in pair-cell recordings. This showed that extrasynaptic excitation in MCs is very weak due to single spikes but rises sharply and supralinearly with increasing spikes, differing from sublinear behavior for synaptic excitation of PG cells. Comparable dynamics leading to a preference for extrasynaptic excitation were also observed during recordings of extrasynaptic and inhibitory currents in response to OSN input of increasing magnitude. The observed alterations in the balance between extrasynaptic excitation and inhibition in glomeruli with stimulus strength could underlie an intraglomerular mechanism for olfactory contrast enhancement. = C77 mV in both cells) used to test the spillover hypothesis. Shown are currents evoked by OSN activation (40 A) in a single response-trial (= 0.0010). Boxed region in shows two examples of current deflections in the MC that were time-locked to quick EPSCs in the PG cell. Open arrowheads in point to bursts of EPSCs in the PG cell that delineate the cell as the subtype that receives direct input from eTCs (Shao et al., 2009). = 0.78, = 0.008). Plot combines data from our standard recordings (= 7; black circles) as well as three recordings in TTx (observe = C77 CYT997 (Lexibulin) mV) evoked by single eTC spikes (black; in LCA mode). Natural traces (left) and averages (= 94) are shown. HBGF-4 Note the amplitude and kinetic similarities to MC currents recorded in the PG cell-MC pairs (Fig. 1= 9) versus PG cell-MC pairs (= 8 for = 7 for reflect mean SEM. Integrated charge values were multiplied by C1. (Fukunaga et al., 2014) studies. Materials and Methods Animals and slice preparation Male and female 8- to 20-d-old Sprague Dawley rats CYT997 (Lexibulin) obtained from Charles River Laboratories were used. All experiments were conducted under protocols approved by the Animal Care and Use Committee of the University or college of Colorado, CYT997 (Lexibulin) Anschutz Medical Campus. Acute horizontal olfactory bulb slices (300C400 m) were prepared following isoflurane anesthesia and decapitation. Olfactory bulbs were rapidly removed and placed in oxygenated (95% O2, 5% CO2) ice-cold answer containing the following: 72 mM sucrose, 83 mM NaCl, 26 mM NaHCO3, 10 mM glucose, 1.25 mM NaH2PO4, 3.5 mM KCl, 3 mM MgCl2, and 0.5 mM CaCl2 adjusted to 295 mOsm. Olfactory bulbs were separated into hemispheres with a razor knife and attached to a stage using adhesive glue applied to the ventral surface of the tissue. Slices were cut using a vibrating microslicer (Leica VT1000S) and were incubated in a holding chamber for 30 min at 32C. Subsequently, the slices were stored at room heat. Electrophysiological recordings Experiments were conducted under an upright Zeiss Axioskop2 FS Plus microscope (Carl Zeiss MicroImaging) fitted with differential interference contrast (DIC) optics, video microscopy and a CCD video camera (Hamamatsu). Recognized cells were visualized with 10 or 40 Zeiss water-immersion objectives. Recordings were performed at 32C35C. The base extracellular recording answer contained the CYT997 (Lexibulin) following: 125 mM NaCl, 25 mM NaHCO3, 1.25 mM NaHPO4, 25 mM glucose, 3 mM KCl, 1 mM MgCl2, and 2 mM CaCl2 (pH 7.3 and adjusted to 295 mOsm), and was oxygenated (95% O2, 5% CO2). The pipette answer for most whole-cell recordings contained the following: 125 mM K-gluconate, 2 mM MgCl2, 0.025 mM CaCl2, 1 mM EGTA, 2 mM Na3ATP, 0.5 mM Na3GTP, and 10 mM HEPES (pH 7.3 with KOH, osmolarity adjusted to 215 mOsm). For whole-cell recordings from eTCs, 30 mM glutamic acid was added to the pipette to prevent run-down of evoked glutamatergic currents (Ma and Lowe, 2007). For whole cell recordings of eTC and MC current responses to OSN activation, the K-gluconate in the pipette answer was replaced with an equimolar amount of cesium methanosulfonate, as well as the sodium channel blocker QX-314 (10 mM) to block action potentials. All whole-cell recordings included 100 M Alexa Fluor 488 or Alexa Fluor 594 in the pipette answer to allow for visualization of cell processes. Loose cell-attached (LCA) recordings from eTCs were made with CYT997 (Lexibulin) a pipette that contained the extracellular answer. Patch pipettes, fabricated.