ARRESTED NEURONAL PROLIFERATION AND IMPAIRED HIPPOCAMPAL FUNCTION FOLLOWING FRACTIONATED BRAIN IRRADIATION IN THE ADULT RAT T. M. MADSEN, a P. E. G. KRISTJANSEN, b T. G. BOLWIG a AND G. WO ¨ RTWEIN a * a Laboratory of Neuropsychiatry, Department of Psychiatry O-6102, H:S Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark b Institute of Molecular Pathology, University of Copenhagen, Copen- hagen, Denmark Abstract—The generation of new neurons in the adult mam- malian brain has been documented in numerous recent re- ports. Studies undertaken so far indicate that adult hip- pocampal neurogenesis is related in a number of ways to hippocampal function. Here, we report that subjecting adult rats to fractionated brain irradiation blocked the formation of new neurons in the dentate gyrus of the hippocampus. At different time points after the termination of the irradiation procedure, the animals were tested in two tests of short-term memory that differ with respect to their dependence on hippocampal function. Eight and 21 days after irradiation, the animals with blocked neu- rogenesis performed poorer than controls in a hippocampus- dependent place-recognition task, indicating that the pres- ence of newly generated neurons may be necessary for the normal function of this brain area. The animals were never impaired in a hippocampus-independent object-recognition task. These results are in line with other reports documenting the functional significance of newly generated neurons in this region. As our irradiation procedure models prophylactic cranial irradiation used in the treatment of different cancers, we suggest that blocked neurogenesis contributes to the reported deleterious side effects of this treatment, consisting of memory impairment, dysphoria and lethargy. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: adult neurogenesis, place recognition, object recognition, working memory, dentate gyrus, granule cell. Adult hippocampal neurogenesis has been documented for a number of years in rodents, and has recently been demonstrated in several other mammalian species, includ- ing humans (Altman and Das, 1965; Eriksson et al., 1998; Kempermann et al., 1997). A number of studies relate increased hippocampal neurogenesis to increased de- mand on hippocampal function. In certain birds, food-stor- ing behavior during the fall is associated with increased neurogenesis (Barnea and Nottebohm, 1994). When rats are trained in tasks, which require an intact hippocampus, there is a greater rate of neurogenesis and increased survival of newborn neurons (Gould et al., 1999; Lemaire et al., 2000). Recently, Shors and colleagues reduced hippocampal neurogenesis in rats to 20% of control levels with an antimitotic agent (Shors et al., 2001). This was associated with impairment of hippocampus-dependent trace conditioning, while leaving hippocampus-indepen- dent delay conditioning unaffected. Newborn dentate granule neurons derive from a pop- ulation of precursor or stem cells, residing in the subgranu- lar zone (Gage et al., 1998). The majority of cells born in this region develop into neurons and migrate into the gran- ule cell layer where they integrate into preexisting neuronal networks, forming connections characteristic of this region (Markakis and Gage, 1999). The time required for a newly born neuron to extend neurites and to attain the charac- teristics of a granule cell is estimated to be around 10 –14 days (Hastings and Gould, 1999). In another study, van Praag and colleagues showed that adult-generated neu- rons, labeled with a green fluorescent protein-retrovirus, display electrophysiological characteristics similar to ma- ture granule cells 28 days after birth, and that these neu- rons have functional synapses at this time (van Praag et al., 2002). Ionizing irradiation has long been known to stop cellu- lar proliferation. At appropriate doses it kills predominantly dividing cells, sparing neighboring, non-dividing cells. In the brains of adult rats, a single 10-Gy (absorbed dose) dose causes apoptosis among proliferating cells in the dentate gyrus of the hippocampus, leaving other cells un- scathed (Peissner et al., 1999). A single 5-Gy dose was reported to block adult neurogenesis (Parent et al., 1999). In the clinic, fractionated whole-brain irradiation is used prophylactically to avoid metastasis to the CNS by eradi- cation of micrometastatic deposits in the brain parenchyma (Auperin et al., 1999). In this study, we examined the effect of fractionated whole-brain irradiation on adult neurogenesis. Our goal was to characterize the consequences of this treatment for hippocampal function. Thus, we compared the perfor- mance of irradiated and non-irradiated rats in two tests of recognition memory, which differ with respect to their de- pendence on hippocampal function (M’Harzi et al., 1991; Steckler et al., 1998). Additionally we examined the ani- mals’ activity level in an open field and their ability to learn the position of a hidden platform in a water maze. It is well established that lesions within the hippocampal formation are associated with changes in both those behaviors (Douglas and Isaacson, 1964; Morris et al., 1982; Walsh et al., 1986). *Corresponding author. Tel: +45-3545-6118; fax: +45-3539-3546. E-mail address: gw6102@rh.dk (G. Wo ¨ rtwein). Abbreviations: BrdU, bromodeoxyuridine; KPBS, potassium phos- phate-buffered saline; KPBS-T, potassium phosphate-buffered sa- line+0.25% Triton X-100; PBS, phosphate-buffered saline. Neuroscience 119 (2003) 635– 642 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00199-4 635