Introduction receptors located on glutamatergic terminals [38,29]. Re-

Brain Research 883 2000 41–50 www.elsevier.com locate bres Research report Stimulation of 5-HT receptors reduces apoptosis after transient 1A forebrain ischemia in the rat Christine Schaper, Yuan Zhu, Maria Kouklei, Carsten Culmsee, Josef Krieglstein ¨ ¨ Institut f ur Pharmakologie und Toxikologie , Fachbereich Pharmazie, Philipps-Universitat Marburg, Ketzerbach 63, D-35032 Marburg, Germany Accepted 15 August 2000 Abstract It has recently been shown that 5-HT receptor stimulation reduced the infarct volume after occlusion of the middle cerebral artery in 1A rats. Since there is increasing evidence that apoptosis is involved in neurodegenerative diseases and stroke, we investigated whether the 5-HT agonist Bay x 3702 could protect neurons against apoptotic damage in a rat model of transient forebrain cerebral ischemia. Bay x 1A 3702 4 mg kg i.v. caused a 10 reduction of neuronal damage in the hippocampal CA1 subfield. Higher doses of Bay x 3702 40 and 12 mg kg i.v. did not cause any neuroprotective effect, most likely because of the strong reduction of mean arterial blood pressure during the period of Bay x 3702 infusion. Bay x 3702 4 mg kg i.v. diminished DNA laddering in the hippocampus and striatum 4 days after 10 min forebrain ischemia. These results were confirmed by TUNEL-staining. The anti-apoptotic effect was abolished by additional treatment with the 5-HT receptor antagonist WAY 100635 1 mg kg. Taken together, the results suggest that Bay x 3702 can rescue 1A hippocampal as well as striatal neurons from apoptotic cell death in vivo via stimulation of 5-HT receptors.  2000 Elsevier Science 1A B.V. All rights reserved. Theme : Disorders of the nervous system Topic : Ischemia Keywords : 5-HT agonist; Forebrain ischemia; WAY 100635; Apoptosis; Neuroprotection 1A 1. Introduction receptors located on glutamatergic terminals [38,29]. Re- cently it has been shown that stimulation of 5-HT 1A It is well known that the stimulation of 5-HT re- receptors inhibits apoptotic cell death in primary cultures 1A ceptors can cause neuroprotection in vitro and in vivo of embryonic chick neurons [3,43] as well as in neuronal [9,36,37,35]. Postsynaptic 5-HT receptors are highly HN-2-5 cells [1]. There is growing evidence that apoptotic 1A expressed in brain regions such as hippocampus or neocor- cell death occurs under pathological conditions, such as tex [34,15]. In these regions the neurons are highly Alzheimer’s disease, Parkinson’s disease and Huntington’s sensitive to damage induced by cerebral ischemia or brain disease [18,30]. It has also been reported that delayed trauma [34,10]. The mechanism which leads to neuro- neuronal death, which is most likely apoptosis, can also protection after activation of the 5-HT receptors is appear after acute insults to CNS, such as cerebral 1A thought to be due to the induction of hyperpolarisation of ischemia [27,28,19]. This demonstration of apoptosis after neuronal membranes [12,14,4]. This effect is most likely cerebral ischemia suggests an increased time window for 1 mediated by opening G protein-coupled K channels. therapeutical intervention [26]. In the light of these data it Additionally, it has been reported that 5-HT receptor is of interest to know whether stimulation of 5-HT 1A 1A agonists can reduce glutamate release [39], which is most receptors could lead to reduced apoptotic cell death in likely mediated through activation of presynaptic 5-HT vivo. Therefore, we used the model of transient forebrain 1A ischemia, where a maximum of apoptotic cells was de- tected at the fourth day after ischemia [51]. For 5-HT 1A Corresponding author. Tel.: 149-6421-282-1311; fax: 149-6421-282- receptor stimulation we used the highly potent 5-HT 8918. 1A E-mail address : krieglstmailer.uni-marburg.de J. Krieglstein. receptor agonist Bay x 3702 2-R-2-[4-[[3,4-dihydro- 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 7 6 - 6 42 C 2H - 1 - benzopyran - 2 - ylmethyl]amino] - butyl] - 1, 2 - ben - removed, the blood was reinfused and NaHCO 50 mg 3 zisothiazol-32H-one 1,1-dioxide monohydrochloride kg, i.v. was administered. Arterial pH, pCO , pO and 2 2 [15,16]. blood pressure were measured before and after ischemia. The aim of this study was to investigate whether the The experiments were approved by the government ethics 5-HT agonist Bay x 3702 could cause an anti-apoptotic committee. All efforts were made to reduce the suffering 1A effect in vivo. In addition, we attempted to find out, of the animals during the experiments. whether this anti-apoptotic effect could be blocked by the selective 5-HT receptor antagonist WAY 100635 N-[2- 2.4. Drug administration 1A [4-methoxyphenyl-piperazinyl]ethyl]-N-2-piperidinyl cy- clohexane carboxamide [20]. Electrophoresis and staining Bay x 3702 40, 12 and 4 mg kg or saline were infused with terminal deoxynucleotidyl transferase TdT-mediated i.v. with a perfusor Secura, Braun, Melsungen, Germany dUTP nick end labeling TUNEL were used to measure immediately after ischemia for a period of 4 h. The DNA laddering and to detect internucleosomal DNA infusion was given into the tail vein with a constant flow fragmentation in situ. rate of 1 ml h. WAY 100635 1 mg kg was injected i.p. 60 min before ischemia. The body temperature was maintained at 3760.58C with a heating lamp during the

2. Material and methods period of infusion and up to 6 h after infusion.