Neurogenesis in the adult rat piriform cortex Anton Pekcec a,b , Wolfgang L˛scher a,b and Heidrun Potschka a,b a Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine and b Center for Systems Neuroscience, Hannover, Germany Correspondence and requests for reprints to Dr H Potschka, Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Buenteweg 17, D-30559 Hannover, Germany Tel: + 49 511 953 8728; fax: + 49 511 953 8581; e-mail: heidrun.potschka@tiho-hannover.de Received 28 January 2006; accepted 31January 2006 Multipotent neural precursors have been suggested to exist in many parts of the adult mammalian brain. In the present study, we characterized the neurogenic potential in the piriform cortex of adult rats. Proliferation rates as detected by 5 0 -bromodeoxy- uridine-labeling proved to be low when compared with the major neurogenic brain regions (i.e. the hippocampus and the subventricular zone). 5 0 -Bromodeoxyuridine/NeuN-labeling in accordance with doublecortin, polysialylated neural cell adhesion molecule, and TUC- 4 -labeling indicated that neuronal di¡erentia- tion of newborn cells occurs predominantly in layer II of the piriform cortex. Many of the cells exhibited a pyramidal cell morphology. The lack of 5 0 -bromodeoxyuridine/NeuN-labeled cells 12 weeks after 5 0 -bromodeoxyuridine administration argued against long-term survival of newborn neurons in the piriform cortex. NeuroReport 17:571^574  c 2006 Lippincott Williams & Wilkins. Keywords: doublecortin, neurogenesis, piriform cortex, polysialylated neural cell adhesion molecule, TUC- 4 Introduction Throughout life, new neurons are generated from progeni- tor cells in specific brain regions of various species, including man [1,2]. A high neurogenic potential has been described for the dentate gyrus of the hippocampus and for the subventricular zone from where newborn cells migrate to the olfactory bulb. In addition, neurogenesis with a low turnover rate has been suggested for other brain regions, including the cortex [3–5]. The piriform cortex is the largest area of the mammalian olfactory cortex that is characterized by direct input from the olfactory bulb via the lateral olfactory tract [6]. With its unique intrinsic associative fiber system and its various connections to and from other limbic structures, the piriform cortex has been implicated in many functional studies [7]. Besides an obvious involvement in olfactory sensory processing, the piriform cortex has gained attention with regard to its potential involvement in memory processing, and in pathological processes such as spread of epileptic seizure activity [7]. As a result of these physiological and pathological functional implications, and owing to the pronounced neurogenesis-associated plasticity in the olfactory bulb, it is of special interest to investigate the neuronal plasticity in the olfactory bulbs’ major output structure, that is, the piriform cortex. In the present study, we characterized the neurogenic potential in the adult rat piriform cortex, including a thorough analysis of the distribution of neuronal progenitor cells within the different layers of the piriform cortex. The existence of neuronal progenitor cells in the piriform cortex was substantiated by immunostaining of various molecular markers of neuronal progenitor cells and by 5 0 -bromo- deoxy-uridine (BrdU)/NeuN double-labeling. Materials and methods Male Sprague–Dawley rats were purchased at a body weight of 200–220 g (Harlan–Winkelmann, Borchen, Ger- many). All experiments were carried out in compliance with the German Animal Welfare Act. All efforts were made to minimize pain or discomfort of the animals used. As a thymidine analog, BrdU is incorporated into the DNA during the S phase of the cell cycle [8]. Each injection of BrdU labels those proliferating cells that are in the DNA- synthetic phase of the cell cycle (S phase). BrdU is available for about 30 min and thus labels a proportion of dividing cells that are in the S phase during this period [9]. Rats received a total of eight intraperitoneal injections of 50 mg/ kg BrdU. The administration was started in the late afternoon. On the next 3 days, the animals received twice- daily administrations in the morning and in the late afternoon with an interval of 8 h. The last BrdU adminis- tration was given in the morning of the fourth day. Rats were killed 2 h later (n¼4). Another group of rats also received eight intraperitoneal injections of 50 mg/kg BrdU, administered in the morning and in the afternoon for 4 consecutive days. This group of rats (n¼4) was killed 12 weeks (84 days) after the last BrdU injection. Following transcardial perfusion of the animals with a fixative, the brains were removed and 40-mm coronal brain sections were cut on a sliding microtome (Frigomobil, Leica, Germany). Cells that incorporated BrdU and expressed the neuronal marker NeuN were identified by immunohistological BrdU/NeuN double-labeling. After washing, pretreatment, and blocking of the sections, they were incubated in an antibody mixture containing rat anti-BrdU (Oxford Biotech- nology, Oxfordshire, UK), 1:20, and anti-NeuN (Chemicon, MOLECULAR NEUROSCIENCE NEUROREPORT 0959-4965  c Lippincott Williams & Wilkins Vol 17 No 6 24 April 2006 571 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.