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Summary:The exceptional ability of the turtle brain to survive prolonged anoxia makes it a unique model for studying anoxic survival mechanisms. We have used epi illumination microscopy to record blood flow rate in ve nules on the cortical surface of turtles (Trachemys scripta). During anoxia, blood flow rate increased 1.7 times after 45-75 min, whereupon it fell back, reaching preanoxic values after 115 min of anoxia. Topical super fusion with adenosine (50 /-LM) during normoxia caused a 3.8-fold increase in flow rate. Superfusing the brain with Freshwater turtles of the genera Trachemys and Chrysemys possess the exceptional ability among vertebrates to tolerate prolonged anoxia, surviving days of anoxia at room temperature and several months at temperatures close to ooe (Ultsch, 1989).In vertebrates, from mammals (Hansen, 1985; Siesjo, 1992) to fish (Nilsson et aI., 1993), the brain is particularly sensitive to anoxia, rapidly losing ATP levels-the primary cause of neuronal death. To maintain brain ATP levels, turtles utilize two cooperating strategies. An initial enhancement of glycolytic ATP production is followed by deep hy pometabolism (Lutz, 1992).An increased glycolytic rate would probably de mand increased cerebral blood flow and/or an ele vated blood glucose level. Previous studies on tur tles using the microsphere technique have shown an approximately threefold increase in cerebral blood Received January 19, 1994; final revision received March 7, 1994; accepted March 8, 1994.Address correspondence and reprint requests to Dr. P.Hylland at Vertebrate Physiology and Behaviour Unit, Depart ment of Limnology, Norbyvagen 20, S-752 36 Uppsala, Sweden. 877the adenosine receptor blocker aminophylline (250 /-LM)totally inhibited the effects of both adenosine and anoxia, while aminophylline had no effect on normoxic flow rate. None of the treatments affected systemic blood pressure. These results indicate an initial adenosine-mediated in crease in cerebral blood flow rate during anoxia, probably representing an emergency response before deep meta bolic depression sets in.

The crucian carp (Carassius carassius) has the rare ability to survive prolonged anoxia, indicating an extraordinary capacity for glycolytic ATP production, especially in a highly energy-consuming organ like the brain. For the brain to be able to increase its glycolytic flux during anoxia and profit from the large liver glycogen store, an increased glucose delivery from the blood would be expected. Nevertheless, the effect of anoxia on brain blood flow in crucian carp has never been studied previously. We have used epireflection microscopy to directly observe and measure blood flow rate on the brain surface (optic lobes) during normoxia and anoxia in crucian carp. We have also examined the possibility that adenosine participates in the regulation of brain blood flow rate in crucian carp. The results showed a 2.16-fold increase in brain blood flow rate during anoxia. A similar increase was seen after topical application of adenosine during normoxia, while adenosine was without effect during anoxia. Moreover, superfusing the brain with the adenosine receptor blocker aminophylline inhibited the effect of anoxia on brain blood flow rate, clearly suggesting a mediatory role of adenosine in the anoxia-induced increase in brain blood flow rate.

Summary: Nitric oxide (NO)-dependent regulation of brain blood flow has not been proven to exist in fish or other ectothermic vertebrates. Using epi-illumination mi croscopy on the brain surface (optic lobes) of crucian carp (Carassius carassius), we show that superfusing the brain with acetylcholine (ACh) induces an increase in ce rebral blood flow velocity that can be completely blocked by the NO synthase inhibitors NG-nitro-L-arginine meth ylester (L-NAME) and NG-nitro-L-arginine. Also, sodium nitroprusside, which decomposes to liberate NO, causes an increase in cerebral blood flow velocity. By contrast,In the mammalian brain, acetylcholine (ACh) causes vasodilation of cerebral arteries, both when it is administered exogenously and when it is re leased from parasympathetic fibers innervating ce rebral arteries and arterioles (Morita-Tsuzuki et aI., 1993). The vasodilatory action of ACh is mediated by an endothelium-derived relaxing factor (EDRF), which is now generally accepted to be identical with nitric oxide (NO) or a closely related molecule (re viewed by Faraci, 1993). Nevertheless, a role for NO in stimulating tissue blood flow by vasodilation in ectothermic vertebrates has not been demon strated. In fact, after having failed to find NO dependent mechanisms in trout vasoregulation, Ol son and Villa (1991) suggested that EDRF-like va sodilator mechanisms are not present in trout vessels.Received March 28,1994; final revision received July 7,1994; accepted August 2, 1994.Address correspondence and reprint requests to Dr. P. Hylland at Vertebrate Physiology and Behaviour Unit, Dept. of Limnology, Uppsala University, Norbyvagen 20, S-752-36 Upp sala, Sweden, Abbreviations used: ACh, acelytcholine; EDRF, endothelium derived relaxing factor; L-NA, NG-nitro-L-arginine; L-NAME, W-nitro-L-arginine methylester; NOS, NO synthase; SNP, so dium nitroprusside, 519L-NAME does not block the increase in blood flow ve locity caused by anoxia. The results suggest that NO is an endogenous vasqdilator in crucian carp brain that medi ates the effects of ACh. Because teleost fish deviated from other vertebrates 400 million years ago, these results suggest that NO-dependent brain blood flow regulation was an early event in vertebrate evolution. Key Words:Adenosine-Aminophylline-Anoxia-Carassius caras sius-Hypoxia-Endothelium-derived relaxing factor NG-Nitro-L-arginine methylester-Sodium nitroprusside.

Summary: Nitric oxide (NO)-dependent regulation of brain blood flow has hitherto not been studied in reptiles. By observing the brain surface (telencephalon) of the freshwater turtle (Trachemys scripta) with epiillumina tion microscopy, we show that topical application of ace tylcholine (ACh) induces an increase in CBF velocity that can be completely blocked by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The effect of L-NAME was reversed by L-arginine. Also, sodium nitroprusside (SNP), which decomposes to liberate NO, caused an increase in CBF velocity. By contrast, Acetylcholine (ACh) causes vasodilation of !pam mali an cerebral arteries, both when administered exogenously and when released from parasympa thetic fibers innervating cerebral arteries and arte rioles (Morita-Tsuzuki et aI., 1993). The vasodila tory action of ACh is mediated by an endothelium derived relaxing factor (EDRF), which is now generally accepted to be identical with nitric oxide (NO) or a closely related molecule (reviewed by Faraci, 1993).Also, hypoxia causes increased brain blood flow in mammals, and the underlying mechanisms have been suggested to include NO production (Kozniewska et al., 1992;Burnstock, 1993). More over, NO is produced in the ischemic mammalian brain (Malinski et aI. , 1993; Sato et aI., 1993), but the role of NO in ischemic brain damage is controReceived March 28, 1995; final revision received June 27, 1995; accepted June 27, 1995. Address correspondence and reprint requests to Dr. P. Hylland at Vertebrate Physiology and Behaviour Unit, Depart ment of Limnology, Norbyvagen 20, S-752 36 Uppsala, Sweden.Abbreviations used: ACh, acetylcholine; EDRF, endothelium derived relaxing factor; L-NAME, N G -nitro-L-arginine methyl ester; NO, nitric oxide; NOS, NO synthase; SNP, sodium nitro prusside. 290L-NAME could not block the increase in blood flow ve locity caused by anoxia. Interestingly, superfusing the brain with ACh or SNP during anoxia had no effect on the blood flow velocity. The results suggest that NO is an endogenous vasodilator in the turtle brain, mediating the effects of ACh during normoxia. By contrast, anoxia does not rely on NO as a vasodilator.

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