Methylxanthine-evoked perturbation of spontaneous and evoked activities in isolated newborn rat hippocampal networks
Introduction
Methylxanthines like caffeine, aminophylline or theophylline are the gold standard for countering apnea of prematurity, but convulsive seizures can occur as a serious side effect (Delanty et al., 1998, Bhatia, 2000, Korematsu et al., 2008, El-Bitar and Boustany, 2009). Such seizures originate from cortico-hippocampal networks that are yet immature at birth (Khazipov and Luhmann, 2006, Ben-Ari et al., 2007, Sipilä and Kaila, 2008, Rakic, 2009). These neural networks show spontaneous discharges, referred to as early network oscillations (ENOs), spindle bursts or gamma oscillations, that are considered as pivotal to consolidate synaptic connectivity, particularly via the associated depolarization-related rises of cytosolic Ca2+ (Goodman and Shatz, 1993, Leinekugel et al., 1997, Canepari et al., 2000, Garaschuk et al., 2000, Ben-Ari et al., 2007, Sipilä and Kaila, 2008, Yang et al., 2009, Yang et al., 2013).
In preterm infants, methylxanthines cause seizures predominantly at blood plasma levels >50 μM (Delanty et al., 1998, Comer et al., 2001, Korematsu et al., 2008, El-Bitar and Boustany, 2009). Although this value is higher than that for impeding adenosine receptors, several of the latter studies consider blockade of tonic neural network inhibition by endogenous adenosine as a major mechanism of such seizures. These reports also point out that therapeutic plasma methylxanthine levels can be as high as 500 μM (Fredholm et al., 1999). As this dose blocks phosphodiesterase-4 (PDE4) (Fredholm et al., 1999), the resulting increase of cellular cyclic-adenosine monophosphate (cAMP) levels may also be epileptogenic. At 1–10 mM, methylxanthines inhibit both A-type γ-aminobutyric acid (GABAA) and glycine receptors as well as sarcoplasmic-endoplasmic reticulum Ca2+ ATPases (SERCAs) (Fredholm et al., 1999). These mechanisms might also play a role in seizures, if methylxanthines accumulate in brain interstitial space to doses higher than in plasma (Ruangkittisakul and Ballanyi, 2010, Panaitescu et al., 2013).
The main objective of our study was to determine threshold concentrations of the frequently used methylxanthines caffeine and theophylline for evoking seizure-like activities in neonatal hippocampal networks and analyze the underlying mechanisms. As a further objective, we studied if these agents affect hippocampal ENOs at doses close to, and slightly above, the common upper limit for therapeutic plasma levels, i.e. 100 and 500 μM, respectively. We also applied low millimolar methylxanthine to assess effects on spontaneous and evoked hippocampal activities caused by GABAA/glycine receptor and SERCA inhibition. For this, we recorded ENOs in the CA3 area (CA3-ENOs) of hippocampal slices (Ben-Ari et al., 1989, Ben-Ari et al., 2007, Sipilä and Kaila, 2008) and electrically evoked CA1 field potentials (CA1-FPs) in intact isolated hippocampi (Khalilov et al., 1997, Kilb et al., 2007) from neonatal rats. In the slices, these electrophysiological population recordings were complemented by imaging of dynamic changes of the free cytosolic Ca2+ concentration in groups of visualized CA3 neurons using multiphoton microscopy (Ruangkittisakul et al., 2008). One aspect of such Ca2+ imaging was to study whether methylxanthine-evoked seizures induce SERCA inhibition-related acute Ca2+ dysregulation that initiates the death of hippocampal neurons in other types of seizures (Griffiths et al., 1984, Pal et al., 2000).
Section snippets
Experimental procedures
ENOs originate from CA3 neurons and trigger synchronous discharges in other hippocampal areas including CA1 (Ben-Ari et al., 2007, Sipilä and Kaila, 2008). Here, we studied methylxanthine effects on CA3-ENOs in slices (Kantor et al., 2012) and monosynaptic CA1-FPs evoked by electrical CA3 stimulation in intact hippocampi which lack spontaneous activity, but comprise an intact neural circuitry (Khalilov et al., 1997, Luhmann and Kilb, 2012). Both in vitro models were isolated from 8 to
Methylxanthine concentrations for evoking seizure-like discharges in slices
We first determined in the slices the doses at which methylxanthines evoke seizure-like discharges, specifically ‘ictal-like events’ (ILEs) and/or ‘interictal-like activities’ (IAs) (Kilb et al., 2007) occurring 2–5 min after start of bath-application of methylxanthine (Fig. 1A–C). The probability of incidence of theophylline-evoked ILEs and/or IAs increased from 8.3% at 0.25 mM and 7.7% at 0.5 mM to 94% at 10 mM (Fig. 1D1). For caffeine, the probability of ILEs and/or IAs increased from 17% at 5 mM
Dose dependence of methylxanthine effects on neural networks
One major finding of our study was that methylxanthine evoked seizure-like discharges in both slices and intact hippocampi primarily at doses ⩾1 mM. In previous studies on ‘silent’ hippocampal and cortical slices from mature rodents, methylxanthine concentrations for evoking (seizure-like) hyperexcitability ranged between low micromolar (Greene et al., 1985, Ault et al., 1987, Dimpfel et al., 1994) and around 1 mM concentrations (Moraidis and Bingmann, 1994, Thöne et al., 2008). This variability
Acknowledgments
Support was provided by Canadian Institutes of Health Research (CIHR), Women’s and Children’s Health Research Institute (WCHRI), Alberta Heritage Foundation for Medical Research (AHFMR), the Canada Foundation for Innovation and Advanced Education and Technology-Alberta (CFI-AET), Deutsche Forschungsgemeinschaft (DFG), German Federal Ministry of Education and Research (KMU-innovativ: Medizintechnik, project TENECOR) and MAIFOR. KB is a AHFMR Scientist.
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Authors contributed equally.