title: Analysis of spontaneous hippocampal activity allows sensitive detection of acetylcholine-mediated effects.
abstract: BACKGROUND
  Excitation of Acetylcholine-mediated (Ach) transmission (especially if irreversible) may pose life-threatening adverse events by increasing neuronal network activity. Unfortunately, adequate detection of this liability during early drug development is hampered, because published ex vivo electrophysiological models are very insensitive to this regard. For example, Eserine, which reversibly inhibits acetylcholinesterase (AchE) in the double digit nM range, affects electrically evoked potentials in hippocampal slices only at ≥10μM. Here, a significantly more sensitive method for detecting Ach-mediated alternations is presented by analyzing spontaneous neuronal network activity in hippocampal slices.
  
  
  METHODS
  The microelectrode array (MEA) technique with an 8×8 electrode grid was applied to analyze evoked and spontaneous extracellular field recordings in parallel from acute rat hippocampal slices. For evoked potentials, the Schaffer collateral CA3-CA1 pathway was electrically stimulated and the resulting field potential analyzed at the CA1 pyramidal layer. Spontaneous spike activity was detected as negative inflections from the 100Hz high pass filtered signal. Spike frequency was analyzed within the whole CA1 region.
  
  
  RESULTS
  Modification of Ach-mediated neuronal transmission via carbachol, Eserine, or Diisopropylfluorophosphate (DFP) does not induce any effects on evoked field potentials at physiologically relevant concentrations. Similar to previous reports, subtle effects were detectable at very high concentrations. By contrast, spontaneous spike frequency was already increased within the expected concentration range. Eserine-induced effects can also be reversed by atropine and washout. On the contrary, effects by the irreversible AchE-blocker DFP could not be washed out.
  
  
  CONCLUSIONS
  Compared to evoked field potentials, spontaneous spike activity in the hippocampal CA1 region appears to be a significantly more sensitive parameter for functional electrophysiological analysis of drug induced Ach-mediated effects. This finding may supplement existing models for detection and prediction of drug-related adverse effects like seizure liability already during early development stages.
authors.affiliation: Merck Serono Research Darmstadt, Merck KGaA, 64293 Darmstadt, Germany. Electronic address: David.Hermann@merckgroup.com.
authors.forename: David
authors.initials: D
authors.lastname: Hermann
authors.affiliation: Merck Serono Research Darmstadt, Merck KGaA, 64293 Darmstadt, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: van Amsterdam
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D008297:Male; D008839:Microelectrodes; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000109:Acetylcholine
keywords: Acetylcholine; Acetylcholinesterase inhibition; Atropine (CID: 174174); Brain slices; Carbachol (CID: 5831); Diisopropylfluorophosphate (PubChem CID: 5936); Eserine (PubChem CID: 5983); Extracellular recordings; Hippocampus; Procaine (PubChem CID: 5795); Safety pharmacology