2. Histamine and the central nervous system GABAergic mechanism in histamine H 3 receptor inhibition of K þ -evoked release of acetylcholine from rat cortex in vivo M. Giorgetti, L. Bacciottini, L. Bianchi, M. G. Giovannini, M. Cecchi and P. Blandina Dipartimento di Farmacologia Preclinica e Clinica, Universita ´ di Firenze, Viale G.B. Morgagni 65, I-50134 Firenze, Italy Introduction Autoradiographic studies have suggested that the presence of H 3 receptors, initially detected as autoreceptors inhibiting histamine release [1], is not restricted to histaminergic neurons [2–4]. Accordingly, functional studies have shown that H 3 receptors modulate the release of several neuro- transmitters, including acetylcholine (ACh) in vitro [5], and in vivo [6]. The present study assessed the location of H 3 receptors modulating ACh release. Materials and methods A transversal microdialysis membrane was implanted in both parietal cortices of male Wistar rats (200–250 g), anesthetized with chloral hydrate (400 mg/kg, i.p.). Twenty-four hours after surgery the membrane was perfused with Ringer solution containing 7 M physostigmine (flow rate: 3 l/min). Ten minute fractions were collected. ACh and GABA contents were measured by HPLC with electrochemical and fluorometric detection, respectively. Rats were stimulated twice by a 10-min exposure to a 100 mM K þ -medium, given through the dialysis fiber at 50 (S 1 ) and 140 min (S 2 ) after equilibration. Spontaneous release values were obtained by averaging ACh and GABA contents in the four samples immediately before S 1 . Drugs were added to the medium 10 min before S 2 and maintained during S 2 , and their effects evaluated by calculating the ratio of the evoked releases (S 2 /S 1 ). All values are expressed as means  SEM, with number of experiments (h). All experiments were done in compliance with the recommendations of the EEC (86/609/CEE) for the care and use of laboratory animals and were approved by the Animal Care Committee of the Universita ´ di Firenze. Results and discussion This study demonstrates that H 3 receptors inhibiting cortical ACh release are not presynaptically located, and facilitate the release of GABA from cortical interneurons. Rat cerebral cortex spontaneously released ACh at stable rates, 4:1  0:6 pmol/10 min (n ¼ 33). Two identical 100 mM K þ stimula- tions (S 1 and S 2 ), each more than doubled ACh release (S 2 /S 1 : 1:33  0:10, n ¼ 6). Tetrodotoxin (0.5 M), a voltage-dependent Na þ -channel blocker, was infused into the prefrontal cortex 20 min before S 2 and maintained during S 2 . K þ -evoked release of ACh failed to show any tetrodotoxin-sensitive component (S 2 /S 1 :1:1  0:1, n ¼ 6), thus excluding involvement of neuronal loops. Imetit (10 M), an H 3 receptor agonist, reduced 100 mM K þ - evoked ACh release in the absence (S 2 /S 1 :0:69  0:01, n ¼ 4), but not in the presence of tetrodotoxin (S 2 /S 1 : 1:1  0:12, n ¼ 4). Hence the suggestion that H 3 receptors modulating ACh release are not located presynaptically on cholinergic nerve terminals, or on non-cholinergic nerve endings impinging on the former. Consistently, H 3 receptor agonists failed to alter [ 3 H]-ACh release from rat cortical synaptosomes [7]. Bicuculline, a GABA A receptor antagonist, reversed the inhibition of ACh release induced by Correspondence to: P. Blandina Fig. 1. The influence of bicuculline on immepip-induced inhibition of 100 mM K þ -evoked release of ACh from cortex of freely moving rats. Bicuculline was infused into the prefrontal cortex through the dialysis fiber 20 min before S 2 and maintained during S 2 stimulation. Immepip was infused, alone or along with bicuculline, 10 min before S 2 and maintained during S 2 . Shown are the means  SEM of 3–6 experiments. ***p < 0:001 vs control by ANOVA and Scheffe’s test. Inflamm. res. 46, Supplement 1 (1997) S33–S34  Birkha ¨user Verlag, Basel, 1997 1023-3830/97/010S33-02 $ 1.50+0.20/0 Inflammation Research 