title: Hyperpolarizing muscarinic responses of freshly dissociated rat hippocampal CA1 neurones.
abstract: 1. Intracellular mechanisms of the muscarinic acetylcholine (ACh) response were investigated in pyramidal neurones freshly dissociated from the rat hippocampal CA1 region. Current recordings were made in the whole-cell mode using the nystatin 'perforated'-patch technique, by which the muscarinic ACh response can be continuously recorded without so-called 'run-down' phenomenon. The amount of intracellular free Ca2+ ([Ca2+]i) was fluorometrically measured using fura-2. 2. In current clamp conditions, ACh induced a transient hyperpolarization accompanied by a decrease in membrane input resistance. 3. Under voltage clamp conditions at a holding potential (Vh) of -40 mV, ACh induced two types of muscarinic currents observed either alone or together: a transient outward current and a slowly activating sustained inward current. 4. The ACh-induced transient outward current reversed the direction at K+ equilibrium potential (EK), and the reversal potential (EACh) shifted 56.7 mV for a tenfold change of extracellular K+ concentration ([K+]o). 5. The ACh-induced transient outward current increased in a sigmoidal fashion with increase in ACh concentration, where the half-maximal concentration (EC50) and the Hill coefficient (n) were 8 x 10(-7) M and 1.9, respectively. Both muscarine and carbamylcholine mimicked the ACh response, but neither McN-A-343 (M1 agonist) nor oxotremorine (cardiac M2 agonist) induced any current. 6. Muscarinic antagonists reversibly blocked the ACh response in a concentration-dependent manner. The inhibitory potency was in the order of atropine > pirenzepine > AF-DX-116. 7. The ACh-induced transient outward current was never recorded when [Ca2+]i was chelated by the acetoxymethyl ester form of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA AM). On the other hand, in Ca(2+)-free external solution containing 2 mM EGTA and 10 mM Mg2+, the ACh response was elicited by the first application and successive ACh applications did not induce any response. Fura-2 imaging showed that [Ca2+]i was increased when ACh was added to the external medium with or without Ca2+, though in Ca(2+)-free medium only the first application of ACh increased the [Ca2+]i. 8. The ACh response was not affected by pretreatment with pertussis toxin (PTX) but the inhibitory effect of ACh on the high-threshold Ca2+ channel was abolished completely. 9. Pretreatment with Li+ enhanced the amplitude of the transient outward current and the increase in [Ca2+]i induced by ACh. 10. The calmodulin antagonists W-7, chlorpromazine and trifluoperazine reversibly inhibited the ACh response in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Neurophysiology, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Wakamori
authors.forename: H
authors.initials: H
authors.lastname: Hidaka
authors.forename: N
authors.initials: N
authors.lastname: Akaike
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives; D005470:Fluorometry; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007473:Ion Channels / Q000187:drug effects; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010277:Parasympathomimetics / Q000494:pharmacology; D037342:Pertussis Toxin; D015221:Potassium Channels / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D007202:Indicators and Reagents; D007473:Ion Channels; D010277:Parasympathomimetics; D015221:Potassium Channels; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D004533:Egtazic Acid; D037342:Pertussis Toxin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D000109:Acetylcholine; D002118:Calcium