EFFECTS OF FRUCTOSE-1,6-BISPHOSPHATE ON MORPHOLOGICAL AND FUNCTIONAL NEURONAL INTEGRITY IN RAT HIPPOCAMPAL SLICES DURING ENERGY DEPRIVATION Y. IZUMI, a * A. M. BENZ, a H. KATSUKI, a M. MATSUKAWA, a D. B. CLIFFORD b AND C. F. ZORUMSKI a a Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA b Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA Abstract—D-Fructose-1,6-bisphosphate, a high energy glyco- lytic intermediate, attenuates ischemic damage in a variety of tissues, including brain. To determine whether D-fructose- 1,6-bisphosphate serves as an alternate energy substrate in the CNS, rat hippocampal slices were treated with D-fructose- 1,6-bisphosphate during glucose deprivation. Unlike pyru- vate, an endproduct of glycolysis, 10 mM D-fructose-1,6- bisphosphate did not preserve synaptic transmission or mor- phological integrity of CA1 pyramidal neurons during glucose deprivation. Moreover, during glucose deprivation, 10-mM D-fructose-1,6-bisphosphate failed to maintain adeno- sine triphosphate levels in slices. D-Fructose-1,6-bisphos- phate, however, attenuated acute neuronal degeneration pro- duced by 200 M iodoacetate, an inhibitor of glycolysis downstream of D-fructose-1,6-bisphosphate. Because (5S, 10R)-()-5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohep- ten-5,10-imine, an antagonist of N-methyl-D-aspartate recep- tors, exhibited similar protection against iodoacetate dam- age, we examined whether (5S, 10R)-()-5-methyl-10, 11-di- hydro-5H-dibenzo [a,d]cyclohepten-5,10-imine and D-fructose-1,6-bisphosphate share a common neuroprotec- tive mechanism. Indeed, D-fructose-1,6-bisphosphate dimin- ished N-methyl-D-aspartate receptor-mediated synaptic re- sponses and partially attenuated neuronal degeneration in- duced by 100-M N-methyl-D-aspartate. Taken together, these results indicate that D-fructose-1,6- bisphosphate is unlikely to serve as an energy substrate in the hippocampus, and that neuroprotective effects of D-fruc- tose-1,6-bisphosphate are mediated by mechanisms other than anaerobic energy supply. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dizocilpine, energy metabolism, fructose- 1,6-bisphospate, ischemia, N-methyl-D-aspartate receptors, pyruvate. D-Fructose-1,6-bisphosphate (FBP), formally called D-fruc- tose-1,6-diphosphate, is a glycolytic intermediate that pro- tects organ systems from lethal injury accompanying shock and ischemia (Markov, 1986). The cardioprotective actions of FBP against acute myocardial infarction are achieved mainly through modulation of anaerobic energy metabolism as a rate-limiting step in glycolysis (Kirtley and McKay, 1977; Hassinen et al., 1991). FBP also reduces injury in the kidneys during hypoxia-ischemia (Didlake et al., 1989). In the CNS, the effects of FBP are not well understood. There is some evidence that FBP attenuates brain damage induced by ischemia (Farias et al., 1990; Karaca et al., 2002), insulin-induced hypoglycemia (Fairas et al., 1989) and cardiogenic shock (Fairas et al., 1986; Zhang et al., 1988). Furthermore, cerebral infarction in rat pups is reduced by FBP (Sola et al., 1996), although FBP failed to protect the hippocampus from ischemic neuronal damage in gerbils and fetal sheep (Tortosa et al., 1993; Fujii et al., 2001). FBP is also reported to provide limited protection of neurons against simulated ischemia in hip- pocampal slices (Liniger et al., 2001). While these neuro- protective effects may result from actions of FBP as a high energy substrate (Fairas et al., 1989), the preservation of ATP levels after hypoxia/ischemia (Gobbel et al., 1994; Espanol et al., 1995) does not necessarily imply that FBP acts as an energy substrate. Rather, there is evidence that exogenous FBP can impair glycolysis by inhibiting phos- phofructokinase (Kelleher et al., 1995), suggesting that the neuroprotective effects are unrelated to metabolic effects. In rat hippocampal slices, glutamate release during hyp- oxia is reduced by FBP and FBP is unable to maintain ATP levels (Bickler and Buck, 1996). Other actions of FBP that could contribute to neuroprotection include calcium chela- tion (Hassinen et al., 1991) and modulation of second messenger systems. To determine whether FBP serves as an alternative energy source to preserve neuronal function, we examined the effects of exogenous FBP on synaptic transmission, ATP levels and neuronal morphology in rat hippocampal slices in the absence of glucose and during neurodegen- eration induced by simulated ischemia and activation of N-methyl-D-aspartate (NMDA) receptors. The hippocam- pal slice preparation is advantageous for determining the properties of agents that act as putative energy sources. We previously used this preparation to demonstrate that pyruvate and lactate preserve functional and moprhologi- cal integrity by serving as energy substrates (Izumi et al., 1994, 1997) and have provided evidence that ketone bod- ies serve as energy sources during neurodevelopment (Izumi et al., 1998). *Corresponding author: Tel: 1-314-747-2987; fax: 1-314-747- 2983. E-mail address: izumiy@psychiatry.wustl.edu (Y. Izumi). Abbreviations: ACSF, artificial cerebrospinal fluid, DNQX, 6,7-dinitroquinoxaline-2,3-dione; EPSP, excitatory postsynaptic potentials, FBP, D-fructose-1,6-bisphosphate; MK801, (5S,10R)-()- 5-methyl-10, 11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine, NMDA, N-methyl-D-aspartate. Neuroscience 116 (2003) 465– 475 0306-4522/03$30.000.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00661-9 465