Abstracts

Hyperventilation is routinely used to lower seizure threshold in the EEG lab. We examined the effects of hypercapnia and hypocapnia on neuronal excitability, pH, and extracellular adenosine concentration in the hippocampal slice preparation. Transverse hippocampal slices were obtained from 6-to 8-week-old Sprague Dawley rats and extracellular field EPSPs (fEPSPs) were recorded from the CA1 region of the stratum radiatum. Bursting in CA3 was induced by tetanic stimulation of the CA3 pyramidal cell layer. The tetanic stimulus consisted of a 100 Hz, 1-second train of stimuli that were of sufficient amplitude to elicit a population spike when delivered at a lower frequency. When bursting was induced by tetanic stimulation, extracellular solutions were modified to: 1.3 mM Ca²⁺, 0.9 mM Mg²⁺ and 3.3 mM K⁺. 2-photon pH imaging was performed by loading slices with BCECF-AM (Molecular Probes, Eugene, Oregon). Slices were incubated in 5-20 [mu]M dye in aCSF plus 0.0133% DMSO, 6.0 x 10^-5 % pluronic acid and 5 mM probenecid. Slices were incubated in dye solution at physiological temperature for at least 30 minutes. Slices were then placed in a recording chamber and perfused normally. Slices were imaged using a Zeiss LSM 510 laser scanning confocal microscope. A Coherent Mira Ti:Sapphire tunable IR laser was used for 2-photon excitation at the pH-sensitive wavelength (795 nM) and fluorescence was detected at the emission wavelength of BCECF-AM (535 nM). An enzymatic sensor was used to measure adenosine release in rat hippocampal slices (Dale et al., 2000) Hypercapnia (P[sub]CO2[/sub] 10%, pH=7.0 or P[sub]CO2[/sub] 20%, pH=6.7) caused adenosine release and adenosine and ATP-dependent inhibition of synaptic transmission in area CA1. This was due to decreased extracellular pH as neither isohydric hypercapnia (P[sub]CO2[/sub] 10%, pH=7.3) nor propionic acid (intracellular acidification alone) caused adenosine release or altered synaptic transmission. Hypocapnia (P[sub]CO2[/sub] 2%, pH=7.7) decreased extracellular adenosine concentrations and increased neuronal excitability via ecto-ATPase, adenosine A[sub]1[/sub]-receptors and ATP P2-receptors. Hypercapnia caused adenosine-dependent attenuation of epileptiform activity in area CA3. Hypocapnia increased the frequency of epileptiform activity via adenosine A[sub]1[/sub]-receptors and ATP P2-receptors. Based on these studies, adenosine and ATP contribute to CO[sub]2[/sub] induced changes in excitability and the pH-dependent conversion of ATP to adenosine via ecto-ATPase represents an important new transduction mechanism for the effects of CO[sub]2[/sub] seizure threshold. (Supported by the NIH (NINDS) and by the Epilepsy Foundation through the generous support of the American Epilepsy Society. The work in the BGF laboratory was supported by The Wellcome Trust, the Anonymous Trust and Tenovus Scotland.)