(C) Bar histograms representing the mean SEM of the increase in glutamate-induced activation by DL-TBOA (10 and 100 M, = 4 and = 4, respectively), t-PDC (10 and 100 M, = 4 and = 5, respectively) or DHK (10 and 100 M, = 4 and = 12, respectively). of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 M) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mgkg?1 i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC. Conclusions and Implications These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus. electrophysiology: brain slice preparation and extracellular recordings Animals were anaesthetized with chloral hydrate (400 mgkg?1 i.p.), and a block of tissue containing the brainstem was rapidly extracted. Coronal slices of 500C600 m thickness containing the LC were cut using a vibratome. The tissue was allowed to recover from the slicing for 90 min in a modified Haas-type interface chamber continuously perfused with artificial CSF (aCSF) at 33C, saturated with 95% O2/5% CO2 (final pH = 7.34), at a flow rate of 1 1.5 mLmin?1. The aCSF contained (in mM): NaCl 126, KCl 3, NaH2PO4 1.25, glucose 10, NaHCO3 25, CaCl2 2 and MgSO4 2. Single-unit extracellular recordings of LC cells were made as described (Mendiguren and Pineda, 2004). The recording electrode was an Omegadot glass micropipette (Sutter Instrument Co., Novato, CA, USA) pulled and filled with NaCl (0.05 M) (tip size of 2C5 m, 3C5 M). The electrode was placed in the Rabbit Polyclonal to EXO1 LC, which was identified visually in the rostral pons as a dark oval area on the lateral borders of the central grey and the (R)-3-Hydroxyisobutyric acid fourth ventricle, just anterior to the genu of the facial nerve. The extracellular signal from the electrode was passed through a high-input impedance amplifier and monitored on an oscilloscope and an audio unit. Individual neuronal (R)-3-Hydroxyisobutyric acid spikes were isolated from the background noise with a window discriminator. The firing rate was analysed by means of a PC-based custom-made programme, which generated histogram bars representing the cumulative number of spikes in consecutive 10 s bins (HFCP?, Cibertec S.A., Madrid, Spain). Noradrenergic cells were identified by their spontaneous and regular discharge activity, the slow firing rate and the long-lasting, positive-negative waveform (Andrade and Aghajanian, 1984). Pharmacological procedures To characterize the functional (R)-3-Hydroxyisobutyric acid role of EAATs in the total glutamate uptake, we tested the excitatory effect of glutamate (0.3 mM, 30 s). To explore the role of EAATs in the re-uptake of synaptically released glutamate, we measured the activation induced by the depolarizing agent KCl (30 mM, 30C60 s) in the presence of the GABAA receptor antagonist picrotoxin (100 M) (Mendiguren and Pineda, 2007). In these assays, the aCSF contained a lower concentration of NaCl, which was equiosmotically substituted for KCl. As described, the duration of the perfusion was (R)-3-Hydroxyisobutyric acid adjusted at the beginning of each experiment to obtain a reproducible effect of glutamate and KCl (Mendiguren and Pineda, 2007; Zamalloa (version 5.0 for Windows, GraphPad Software, Inc., San Diego, CA, USA). Data are given as (R)-3-Hydroxyisobutyric acid mean SEM. Statistical evaluation was carried out by a paired Student’s < 0.05. Drugs and reagents The following drugs were purchased from Tocris Bioscience (Bristol, UK): alphaxalone, D-AP5, 8-chloro-2-methyl-11H-imidazo[1,2-c][2,3]benzodiazepin-6-benzeneamine dihydrochloride (GYKI 52466), CNQX, DHK, RS-MCPG, nicergoline, DL-TBOA and t-PDC. The following drugs were purchased.