Journal of Cerebral Blood Flow and Metabolism 12:103-109 © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York
Changes in Brain Monoaminergic Neurotransmitter Concentrations in Rat After Intracerebroventricular Injection of Streptozotocin
Andreas Ding, Roger Nitsch, and Siegfried Hoyer Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Heidelberg, Germany
Summary: The tissue concentrations of the monoaminer gic neurotransmitters noradrenaline (NA), dopamine, and serotonin (5-HT) and of their major metabolites were measured by HPLC and electrochemical detection in sev eral rat brain areas after intracerebroventricular injection of streptozotocin (STZ). NA levels were found to be de creased in the frontal cortex by 14%, in the entorhinal cortex by 18%, and in the striatum by 38%. In the ento rhinal cortex, 5-HT levels were decreased by 19% and the 5-HT turnover rate, measured as the 5-hydroxyindoleace-
tic acid/5-HT ratio, was found to be increased by 48%. These results may be indicative of a distinct susceptibility of some neurotransmitters in certain brain areas after a more general impairment of brain metabolism by means of intracerebroventricular application of the diabetogenic compound STZ. Key Words: Brain glucose metabolism Brain insulin-High performance liquid chromatogra phy-Monoaminergic neurotransmitters-Streptozo tocin.
The energy metabolism in the central nervous system and thus normal neuronal function and structure are based on the breakdown of glucose, which is the major fuel for biological energy (Sokoloff, 1977, 1980; Siesj6, 1978). In the nonneu ral tissues, glycolytic glucose breakdown and pyru vate oxidation are known to be controlled by insulin and insulin receptors, respectively (Kahn, 1985), but it is not yet clear whether insulin and the insulin receptors have the same functions also in the ner vous tissue. The existence of both insulin mRNA (Young, 1986; Marks et aI., 1990) and insulin recep tors in brain has already been established (Havrank ova et aI., 1978; Hill et aI., 1986; Werther et aI.,
1987). There is evidence that the concentrations of both insulin and insulin receptors in the brain are independent of peripheral insulin levels (Havrank ova et aI., 1979). However, their functions are still the subject of controversy (for review see Baskin et al. , 1987). Several investigations demonstrate an important role of insulin in the energy metabolism of neurons as in other cells (Phillips and Coxon, 1976; Iguchi et aI., 1981; Amir and Shechter, 1987; Pellegrino et aI., 1987). Moreover, insulin has been found to be involved in neurotransmission. The synaptic specific reuptake of noradrenaline (NA) is inhibited by insulin, indicating a neuromodulatory action of insulin in the brain (Boyd et aI., 1985). Insulin inhibits the firing rate of hippocampal pyra midal neurons (Palovcik et aI., 1984) and of sympa thetic nerves (Sakaguchi and Bray, 1987) and stim ulates the maturation of cholinergic neurotransmis sion (Puro and Agardh, 1984). Streptozotocin (STZ) is established as a means of inducing experimental diabetes mellitus in rats when given by intraperitoneal administration (Junod et aI., 1968; Mordes and Rossini, 1981); this is achieved by way of inhibition of the insulin syn thesis in pancreatic islet cells (Bolaffi et aI., 1987; Zucker and Archer, 1988), decreased autophospho-
Received January 31, 1991; revised May 6, 1991; accepted May 23, 1991. Address correspondence and reprint requests to Prof. Dr. med. S. Hoyer at Department of Pathochemistry and General Neurochemistry, University of Heidelberg, 1m Neuenheimer Feld 220-221, D-6900 Heidelberg, Germany. Dr. A. Ding's present address is Mass Spectrometry Facility, University of California, San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0446, U.S.A. Abbreviations used: DA, dopamine; DOPAC, 3,4,-dihydroxy phenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; icv, intracerebroventricular; NA, noradrenaline; STZ, streptozotocin.
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rylation of the insulin receptor kinase (Kadowaki et aI., 1984), induced insulin resistance (Hussin and Skett, 1988), and also suppression of an insulin responsive glucose transporter (Garvey et aI., 1989). When applied to the brain, STZ may there fore be expected to interfere either with insulin syn thesis or with insulin receptor function or both. Provided brain insulin acts in the same manner as it does in nonnervous tissues (Kahn, 1985), an im pairment of the brain insulin/insulin receptor sys tem is thus likely to cause disturbances in cerebral glucose and related metabolism. Intracerebroven tricular (icv) administration of STZ in a subdiabe togenic dose did, in fact, result in decreased utili zation of glucose and increased release of lactate from the rat brain (Nitsch et aI., 1989), as well as in reduced concentrations of both ATP and creatine phosphate in rat brain cortex (Nitsch and Hoyer, 1991). The same treatment leads to an impairment of locomotor activity and of learning and memory in rats monitored by different psychometric tests (Mayer et aI., 1989, 1990). This may be due to ab normalities in the cholinergic system, in which nerve growth factor and choline acetyltransferase levels are altered in the septal region and the cortex subsequent to icv STZ administration, via de pressed cerebral glucose metabolism (Hellweg et aI., 1989). There are at least two different conditions In which cerebral glucose utilization is found to be preferentially perturbed in the presence of normo glycemia: aging (Gage et aI., 1984; Smith, 1984; Dastur, 1985; Hoyer, 1990) and incipient dementia of the Alzheimer type (DeLeon et aI., 1983; Hoyer et aI., 1988, 1991). The latter is characterized by an imbalance between (reduced) glucose utilization and (fairly normal or only slightly diminished) oxy gen consumption. There is evidence that the pertur bation in cerebral glucose metabolism precedes changes in glucose-related metabolic pathways (Hoyer, 1988; Hoyer and Nitsch, 1988), such as the metabolism of amino acids, which are assumed to be partly used to substitute for lacking glucose as a fuel for energy formation (Hoyer and Nitsch, 1989). ATP production (measured and calculated) was found to be reduced in the brain of patients suffer ing from incipient dementia of the Alzheimer type (Sims et aI., 1983; Hoyer, 1991). This energy deficit of �20% may obviously give rise to subsequent ab normalities in energy-dependent metabolic pro cesses (Siesjo, 1981; Erecinska and Silver, 1989), one of which is the storage and turnover of mono aminergic neurotransmitters (Tissari et aI., 1969; Mandel et aI., 1975). In brain affected by dementia of the Alzheimer type, alterations were found in the
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concentrations of the monoaminergic neurotrans mitters NA, dopamine (DA), and serotonin (5-HT), that of 5-HT being the most markedly reduced after acetylcholine (e.g., Gottfries et aI., 1983; Palmer et al., 1987, 1988; Reinikainen et al., 1988; Herregodts et aI., 1989). Therefore, the present study was designed to in vestigate whether or not the metabolism of the monoaminergic neurotransmitters NA, DA, and 5-HT changes subsequent to icv STZ injection and, if so, which neurotransmitters are involved and in which brain areas. It was also of interest to learn whether or not a rather general variation in mono aminergic neurotransmitters occurred or whether the latter changed more specifically. Here we demonstrate that NA drops markedly in the striatum and in the frontal and entorhinal corti ces; 5-HT drops most markedly in the entorhinal cortex with a concomitant increase in its turnover rate; and DA is unchanged in all brain areas inves tigated. Thus, a disturbance in cerebral glucose/ energy metabolism may precede abnormalities in distinct monoaminergic neurotransmitter concen trations in distinct brain areas.
MATERIALS AND METHODS Animals
One-year-old male Wistar rats weighing between 390 and 520 g were obtained from the Zentralinstitut fUr Ver suchstierzucht (Hannover, F. R.G.). They were housed two per cage with free access to water and food pellets (Altromin standard, no. 1320). An inverse day/night cycle ( 12 h/ 12 h) allowed the performance of experiments dur ing the active period of the circadian cycle. The animals were divided randomly into two groups for icv injection of either STZ (n 17) or artificial CSF (n 17). =
=
Surgical procedure
After anesthesia with chloral hydrate (240 mg/kg body wt i.p. in a 4% solution, Riedel-de Haen, Seelze, F. R. G.), each animal received a single unilateral (left side) icv in jection while held in a stereotaxic apparatus (Uhl, Asslar, F.R.G.). In the first group, STZ (Sigma, Munich, F. R. G. ) was injected in a subdiabetogenic dose ( 1. 5 mg/kg body wt). In the second group, which served as the control group, artificial CSF ( 120 mM NaCi, 3 mM KCl, 1. 15 mM CaCI2, 0.8 mM MgCi2, 27 mM NaHC03, and 0. 33 mM NaH2P04, pH adjusted to 7.2 by CO2 insufflation (all chemicals from Merck, Darmstadt, F. R.G.) was injected. The volume of fluid injected was the same in both groups (9 ILl). Three weeks after the icv injection, the animals of both groups were killed by freezing of their brains in situ with liquid nitrogen under steady-state conditions (anesthesia 0. 5 vol% halothane, nitrous oxide/oxygen 70:30) in arte rial normotension, normocapnia, normoxemia, normogly cemia, and normothermia and then stored at - 80°C until preparation of the different brain areas.
BRAIN MONOAMINE METABOLISM AFTER STREPTOZOTOCIN Sample preparation
Samples from the frontal cortex (43.2 ± 15.8 mg), the temporal cortex (48.0 ± 18.2 mg), the entorhinal cortex (36.9 ± 13. 1 mg), the anterior part of the hippocampus (47.1 ± 21.8 mg), and the dorsolateral striatum ( 1 1 1.2 ± 37.8 mg) were dissected from horizontal brain slices ( 1.5 mm thick) in a glove box maintained at - lYC. After weighing, the frozen brain samples were homogenized with a potter and sonicated for 1 min in 0.7 ml of an ice-cold HPLC buffer as described below. After centrif ugation at 50,000 g for 20 min and extraction with a 2-fold volume of n-heptane (Merck) to remove cell fragments, denatured proteins, and lipids, the aqueous phase was filtered through a OA5-f1m Millipore membrane filter, af ter which 10-30 f11 of each filtrate was injected directly into the chromatographic system, with exception of the filtrates from the striatum samples; these were diluted with a lO-fold volume of the HPLC buffer for measure ment of DA and 3 , 4-dihydroxyphenylacetic acid (DOPAC) levels. Chromatographic system
The HPLC system was made up of a Gilson 23 1140 1 autoinjector (Abimed, Langenfeld, F.R.G.), a Series 1 LC-pump (Perkin Elmer, Uberlingen, F.R.G.), a pulse damper (Biotronik, Maintal, F.R.G.), a precolumn, 20 x 4.6 mm, packed with Nucleosil NH2 5 m, an analytical column, 250 x 4.0 mm, packed with Nucleosil 100-5C18 (Macherey-Nagel, Duren, F.R.G.), and a Coulochem ESA detector (Environmental Sciences Assoc., Bedford, MA, U.S.A.) with a model 50 1 1 analytical cell. The de tector potentials were as follows: detector 1: + 0.32 V (oxidation); detector 2: -0040 V (reduction). The column temperature was controlled by means of a glass jacket coupled to a water thermostat (Colora, Lorch, F.R.G.) maintained at 28°C. The sampling and evaluation of data were carried out with Nelson 2600 chromatography soft ware, run on an IBM-compatible personal computer and a Nelson interface (ESWE, Sinsheim, F.R.G.). Separation of the biogenic amines NA, DA, DOPAC, homovanillic acid (HVA), 5-HT, and 5-hydroxyindole acetic acid (5-HIAA) was obtained with a 0. 1 M phos phate buffer (NaH2PO4 H20; Merck), pH 3. 1, contain ing 3 mM I-octane sulfonic acid Na salt (Janssen, Jeel, Belgium), 0.5 mM disodium ethylenediaminetetraacetate (Merck), 1.5 mM NaN3 (Ferak, Berlin, F.R.G.), and 24 vol% methanol (Merck) within 18 min. All chemicals were of analytical grade. Standards were obtained from Serva (Heidelberg, F.R.G.). •
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± 0.06 ng/mg wet wt in the entorhinal cortex, and 0. 34 ± 0.15 ng/mg wet wt in the striatum (Fig. O. STZ icv resulted in a significant decrease in the NA levels in the striatum (0.21 ± 0.06 ng/mg wet wt), in the entorhinal cortex (0.28 ± 0.05 ng/mg wet wt), and in the frontal cortex (0.38 ± 0. 04 ng/mg wet wt) (see Fig. 1). In the temporal cortex and the hippo campus, no differences from the control groups were observed.
5-HT tissue concentrations
The 5-HT levels in the frontal cortex (0.79 ± 0.17 ng/mg wet wt) and in the striatum (0.80 ± 0.22 ng/ mg wet wt) were about twice as high as in the other brain areas investigated (0.34 ± 0. 10 ng/mg wet wt in the hippocampus, 0.40 ± 0.14 ng/mg wet wt in the temporal cortex, and 0.43 ± 0.08 ng/mg wet wt in the entorhinal cortex in the control group) (Fig. 2). A significant difference between the icv STZ treated group and the control group was observed in the entorhinal cortex, where the 5-HT tissue con centration decreased to 0. 35 ng/mg wet wt (Fig. 2). 5-HIAA tissue concentration and 5-HT turnover rate
The 5-HIAA levels were highest in the striatum (0.86 ± 0.15 ng/mg wet wt) and in the frontal cortex (0.67 ± 0. 12 ng/mg wet wt) of the control group (Fig. 3). In the hippocampus (0.42 ± 0.09 ng/mg wet wt), the entorhinal cortex (0.37 ± 0.08 ng/mg wet wt), and the temporal cortex (0.28 ± 0.05 ng/mg wet wt), the 5-HIAA levels were lower, as were the 5-HT levels. STZ icv had no effect on the 5-HIAA tissue concentrations (Fig. 3). The 5-HT turnover rate, which was calculated as the 5-HIAA/5-HT ra tio, was found to be elevated from 0. 82 ± 0.26 (con trol) to 1. 21 ± 0.47 (STZ) in the entorhinal cortex (Fig. 4). No further changes were observed. 0.8 c::::J control c::::J STZ ICV 0.6 " �
Statistical analysis
Data are expressed as means ± SD (n 17). They were analyzed by means of the nonparametric two-tailed Mann-Whitney U test. Differences were accepted as sta tistically significant at p < 0.05. =
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RESULTS NA tissue concentrations
In the control group, NA levels were highest in the frontal cortex (0.44 ± 0.06 ng/mg wet wt), the mean values being lower in all other areas studied: 0.29 ± 0.06 ng/mg wet wt in the temporal cortex, 0.32 ± 0.08 ng/mg wet wt in the hippocampus, 0.34
CF
CT
CE
ST
HC
FIG. 1. Noradrenaline (NA) tissue concentrations (mean
±
SO, ng/mg wet wt) in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intracerebroventricu lar streptozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cortex; ST, striatum; HC, hippocampus. *p < 0.05 versus corresponding control group.
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'Aj 1.2
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FIG. 2. Serotonin (5-HT) tissue concentrations (mean
CF
±
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ng/mg wet wt) in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intracerebroventricular strep tozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cortex; ST, striatum; HC, hip pocampus. 'p < 0.05 versus corresponding control group.
Tissue concentrations of DA, DOPAC, and HVA; DA turnover rate
The DA levels in the different brain areas ranged from values near the detection limit in the temporal and entorhinal cortex (both 0.09 ± 0.02 ng/mg wet wt) and the hippocampus (0.06 ± 0.01 ng/mg wet wt), through moderate values in the frontal cortex (0.32 ± 0.09 ng/mg wet wt), to high values in the striatum (7.12 ± 1.11 ng/mg wet wt). The DA me tabolites were detected only in the striatum (DOPAC: 1.42 ± 0.34 ng/mg wet wt; HVA: 0.94 ± 0.20 ng/mg wet wt). No differences were found be tween the control group and the STZ group. The DA turnover rate in the striatum (0.29 ± 0.03), cal culated as DOPAC + HVA/DA, also did not differ significantly between these two groups. DISCUSSION
The results obtained in this study demonstrate an effect of icv STZ administration on the tissue con-
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o control EZ2I 5TZ icy
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FIG. 3. 5-Hydroxyindoleacetic acid (5-HIAA) tissue concen trations (mean ± SO, ng/mg wet wt) in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intra cerebroventricular streptozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cor tex; ST, striatum; HC, hippocampus. No statistically signifi cant differences between the groups. J Cereb Blood Flow Metab, Vol.12, No.1, 1992
CT
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I
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FIG. 4. Serotonin (5-HT) turnover rate (mean
HC
SO) calcu lated as 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intracerebroventricular streptozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cortex; ST, striatum; HC, hippocampus. 'p < 0.05 versus corresponding control group. ±
centrations of the monoaminergic neurotransmit ters NA and 5-HT in distinct brain areas. Under the experimental conditions studied, both the N A (-18%) and the 5-HT (-19%) concentrations were reduced to a similar extent in the entorhinal cortex. In the frontal cortex, only the NA tissue concentra tion was reduced, by 14%. Whereas these two neu rotransmitter systems are shown to be susceptible, the dopaminergic system does not seem to be in volved in these damaging mechanisms. This fact be came particularly obvious in the striatum, where the DA level was unaffected and the NA level was decreased by 38%. No changes were observed in the temporal cortex or in the hippocampus. The relationship between (pancreas) insulin effi cacy and brain neurotransmitters has been investi gated after the induction of experimental diabetes mellitus in rats in various studies. Th� most fre quent result reported has been a reduction in the cerebral tissue concentration of the 5-HT precursor tryptophan by 30--50% observed 1-4 weeks after intraperitoneal injection of STZ (Curzon and Fernando, 1977; Mackenzie and Trulson, 1978a; Trulson et aI., 1986). In contrast to these reports, according to which there were no alterations in the brain tissue levels of 5-HT or 5-HIAA, concentra tions of these were also reduced 2 weeks after in traperitoneal STZ treatment in the study of Kwok and Juorio (1987). In addition, a diminished synthe sis and turnover rate of cerebral 5-HT were found in rats with STZ-induced diabetes 2-3 weeks (Crandall et aI., 1981) and 4-6 weeks (Trulson et aI., 1986) after the injection. All the diabetes mellitus-related changes in brain referred to here were reported to be reversible after treatment with insulin. Besides 5-HT, the DA syn thesis in the striatum and the limbic forebrain was also found to be reduced (Trulson and Himmel,
BRAIN MONOAMINE METABOLISM AFTER STREPTOZOTOCIN 1985), as was the DA turnover rate in the striatum (Shimomura et aI., 1988). The turnover rate of NA correlated positively with the peripheral concentra tion of insulin (Smythe et aI., 1984; Trulson and Himmel, 1985). Thus, all monoaminergic neuro transmitter systems in the brain seem to be suscep tible to impairment during diabetes mellitus induced by intraperitoneal injection of STZ. Otherwise, when the immediate effect of insulin on the serotonergic metabolism of the brain was studied, icv administration of insulin was found to have no effect on the cerebral concentrations of tryptophan, 5-HT, or 5-HIAA (MacKenzie and Trulson, 1978a,b). When STZ was injected icv, Lackovic and Salkovic (1990) found an increase in the rat brain tissue levels of NA, DA, and 5-HT 1 week afterward. This result seems to be in contrast to our findings in this study. It must, however, be borne in mind that Lackovic and Salkovic (1990) pooled the whole brain for their studies and did not separate distinct brain areas from one another. Fur thermore, they used a different analytical tech nique, which was not clearly described in their ar ticle. In accordance with most of the aforemen tioned investigations, the data from this study show a relationship between insulin efficacy and mono aminergic neurotransmitter levels in the brain, in particular a reduction of 5-HT and NA in several brain areas. Assuming that insulin acts in the brain in the same way as in nonnervous tissues, our data suggest a direct influence of insulin on neuronal glu cose metabolism and thus on subsequent energy dependent events such as neurotransmitter metab olism in general (Erecinska and Silver, 1989) and storage/packing (Mandel et aI., 1975) and turnover/ reuptake (Tissari et aI., 1969) of neurotransmitters in particular. The specific pattern of the alterations may point to a selective vulnerability of the inner vated target cells. However, the cause of this re mains obscure. We speculate that the density of insulin receptors, which is known to be highest in the olfactory and limbic areas (Hill et aI., 1986; Werther et aI., 1987), may play a role in the inci dence of metabolic changes. The entorhinal cortex, which is a part of the limbic structures, is regarded as one of the most vulnerable brain areas as was demonstrated for dementia of the Alzheimer type (Hyman et aI., 1984). From a functional point of view, this area is of importance with respect to cog nitive processes: Entorhinal neurons carry afferent glutamatergic fibers, which provide the major cor tical input to the hippocampus via the perforant pathway (Hyman et aI., 1986, 1987). As detailed above, this study demonstrated disturbances in neurotransmitter metabolism in this brain area. The observed deficits in learning and memory
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(Mayer et aI., 1989, 1990) may also be at least partly related to the lesions of the serotonergic and nor adrenergic system. In particular, the serotonergic system is thought to be involved in cognitive pro cesses (Wenk et aI., 1987), whereas the noradren ergic system seems to be of minor significance in this respect (Gold and Welsh, 1987; Altman and Normile, 1988; Pontecorvo et aI., 1988). In conclu sion, we have demonstrated in this study that se lective losses in the tissue concentrations of the monoaminergic neurotransmitters NA and 5-HT may be due to an impairment of the cerebral glucose and energy metabolism after icv administration of STZ. Although it is not yet clear whether these al terations are the cause or the consequence of dam aging processes in some of their target regions, they are thought to be a metabolic basis of the deficits in learning and memory following intracerebral STZ application. Acknowledgment: We thank Roland Galmbacher, Gunter Galmbacher, Vera Apell, and Marina Traschutz for expert technical assistance and Jutta-Maria Herb, Janet Dodsworth, and Gunter Mayer for their helpful dis cussions of the manuscript. A. Ding was stipendiary of the Hirnliga e. V. Heidelberg (F.R. G. ).
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Mandel P, Mack G, Goridis C (1975) Function of the central catecholaminergic neuron: synthesis, release, and inactiva tion of transmitter. In: Catecholamines and Behavior, Vol]: Basic Neurobiology (Friedhoff AJ, ed), New York, Plenum Press pp 1-40 Marks JL, Porte D, Stahl WL, Baskin DG (1990) Localization of insulin receptor mRNA in rat brain by in situ hybridization. Endocrinology 127:3234-3236
Mayer G, Nitsch R, Hoyer S (1989) Impairments in passive avoidance learning after cerebral inhibition of cerebral glu cose metabolism. J Neural Transm (P-D Sect) I: 103-104 Mayer G, Nitsch R, Hoyer S (1990) Effects of changes in pe ripheral and cerebral glucose metabolism on locomotor ac tivity, learning and memory in adult male rats. Brain Res 532:95-100
Mordes JP, Rossini RA (1981) Animal models of diabetes. Am J Med 70:353-360
Nitsch R, Hoyer S (1991) Local action of the diabetogenic drug, streptozotocin, on glucose and energy metabolism in rat brain cortex. Neurosci Lett 128:199-202 Nitsch R, Mayer G, Hoyer S (1989) The intracerebroventricu larly streptozotocin-treated rat: impairment of cerebral glu cose metabolism resembles the alterations of carbohydrate metabolism of the brain in Alzheimer's disease. J Neural Transm (P-D Sect) 1:109-110 Palmer AM, Francis PT, Bowen DM, Benton JS, Neary D, Mann DMA, Snowden JS (1987) Catecholaminergic neurons assessed ante-mortem in Alzheimer's disease. Brain Res 414:365-375
Palmer AM, Stratmann GC, Procter AW, Bowen DM (1988) Pos sible neurotransmitter basis of behavioral changes in Alz heimer's disease. Ann Neurol 23:616-620 Palovcik RA, Phillips MI, Kappy MS, Raizada MK (1984) Insu lin inhibits pyramidal neurons in hippocampal slices. Brain Res 309:187-191
Pellegrino DA, Miletich DJ, Albrecht RF (1987) Effect of super fused insulin on cerebral cortical glucose utilization in awake goats. Am J Physiol 253:E4I8-E427 Phillips ME, Coxon RV (1976) Effect of insulin and phenobarbi tal on uptake of 2-deoxyglucose by brain slices and hemi diaphragms. J Neurochem 27:643-645 Pontecorvo MJ, Clissold DB, Conti LH (1988) Age-related cog nitive impairments as assessed with an automated repeated measures memory task: implications for the possible role of acetylcholine and norepinephrine in memory dysfuliction. Neurobiol Aging 9:617-625
Puro DG, Agardh E (1984) Insulin-mediated regulation of neu ronal maturation. Science 225:1170-1172 Reinikainen KJ, Paljarvi L, Hauskonen M, Soininen H, Laakso M, Riekkinen PJ (1988) A postmortem study of noradrener-
BRAIN MONOAMINE METABOLISM AFTER STREPTOZOTOCIN gic, serotonergic and GABAergic neurons in Alzheimer's disease. J Neurol Sci 84: 101-116 Sakaguchi T, Bray GA (1987) Intrahypothalamic injection of in sulin decreases firing rate of sympathetic nerves. Proc Natl Acad Sci USA 84:2012-2014 Shimomura Y, Shimizu H, Takahashi M, Sato N, Uehara Y, Suwa K, Kobayashi I, Tadokoro S, Kobayashi S (1988) Changes in ambulatory activity and dopamine turnover in streptozotocin-induced diabetic rats. Endocrinology 123:2621-2625 Siesjo BK, ed (1978) Brain Energy Metabolism. Chichester, Wiley, pp 151-209 Siesjo BK (1981) Cell damage in the brain: a speculative synthe sis. J Cereb Blood Flow Metab 1:155-185 Sims NR, Bowen DM, Neary D, Davison AN (1983) Metabolic processes in Alzheimer's disease: adenine nucleotide con tent and production of 14 C02 from [U-14C]glucose in vitro in human neocortex. J Neurochem 41:1329-1334 Smith GB (1984) Aging and changes in cerebral energy metabo lism. Trends Neurosci 7: 203-208 Smythe GA, Grunstein HS, Bradshaw JE, Nicholson MY, Compton PJ (1984) Relationship between brain noradrener gic activity and blood glucose. Nature 308:65...{i7 Sokoloff L (1977) Relation between physiological function and energy metabolism in the central nervous system. J Neuro chern 29: 13-26 Sokoloff L (1980) The relationship between function and energy
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metabolism: its use in the localisation of functional activity in the nervous system. Neurosci Res Progr Bull 19: 159210 Tissari AH, Schonhofer PS, Bogdanski F, Brodie BB (1969) Mechanism of biogenic amine transport. II. Relationship be tween sodium and the mechanism of ouabain blockade and the accumulation of serotonin and norepinephrine by synap tosomes. Mol Pharmacol 5: 593--604 Trulson ME, Himmel CD (1985) Effects of insulin and strepto zotocin-induced diabetes on brain norepinephrine metabo lism in rats. J Neurochem 44: 1873-1876 Trulson ME, Jakoby JH, MacKenzie RG (1986) Streptozotocin induced diabetes reduces brain serotonin synthesis in rats. J Neurochem 46: 1068-1072 Wenk G, Hughey D, Boundy Y, Kim A, Walker L, Olton D (1987) Neurotransmitters and memory: role of cholinergic, serotonergic, and noradrenergic systems. Behav Neurosci 101:325-332 Werther GA, Hogg A, Oldfield BJ, McKinley MJ, Figdor R, Allen AM, Mendelsohn FAO (1987) Localization and char acterization of insulin receptors in rat brain and pituitary gland using in vitro autoradiography and computerized den sitometry. Endocrinology 121: 1562-1570 Young WS (1986) Periventricular hypothalamic cells in the rat brain contain insulin mRNA. Neuropeptides 8:93-97 Zucker PF, Archer MC (1988) Streptozotocin toxicity to cultured pancreatic islets of the Syrian hamster. Cell Bioi Toxicol 4:349-356
J Cereb Blood Flow Metab, Vol. 12, No. 1, 1992
Changes in Brain Monoaminergic Neurotransmitter Concentrations in Rat After Intracerebroventricular Injection of Streptozotocin
Andreas Ding, Roger Nitsch, and Siegfried Hoyer Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Heidelberg, Germany
Summary: The tissue concentrations of the monoaminer gic neurotransmitters noradrenaline (NA), dopamine, and serotonin (5-HT) and of their major metabolites were measured by HPLC and electrochemical detection in sev eral rat brain areas after intracerebroventricular injection of streptozotocin (STZ). NA levels were found to be de creased in the frontal cortex by 14%, in the entorhinal cortex by 18%, and in the striatum by 38%. In the ento rhinal cortex, 5-HT levels were decreased by 19% and the 5-HT turnover rate, measured as the 5-hydroxyindoleace-
tic acid/5-HT ratio, was found to be increased by 48%. These results may be indicative of a distinct susceptibility of some neurotransmitters in certain brain areas after a more general impairment of brain metabolism by means of intracerebroventricular application of the diabetogenic compound STZ. Key Words: Brain glucose metabolism Brain insulin-High performance liquid chromatogra phy-Monoaminergic neurotransmitters-Streptozo tocin.
The energy metabolism in the central nervous system and thus normal neuronal function and structure are based on the breakdown of glucose, which is the major fuel for biological energy (Sokoloff, 1977, 1980; Siesj6, 1978). In the nonneu ral tissues, glycolytic glucose breakdown and pyru vate oxidation are known to be controlled by insulin and insulin receptors, respectively (Kahn, 1985), but it is not yet clear whether insulin and the insulin receptors have the same functions also in the ner vous tissue. The existence of both insulin mRNA (Young, 1986; Marks et aI., 1990) and insulin recep tors in brain has already been established (Havrank ova et aI., 1978; Hill et aI., 1986; Werther et aI.,
1987). There is evidence that the concentrations of both insulin and insulin receptors in the brain are independent of peripheral insulin levels (Havrank ova et aI., 1979). However, their functions are still the subject of controversy (for review see Baskin et al. , 1987). Several investigations demonstrate an important role of insulin in the energy metabolism of neurons as in other cells (Phillips and Coxon, 1976; Iguchi et aI., 1981; Amir and Shechter, 1987; Pellegrino et aI., 1987). Moreover, insulin has been found to be involved in neurotransmission. The synaptic specific reuptake of noradrenaline (NA) is inhibited by insulin, indicating a neuromodulatory action of insulin in the brain (Boyd et aI., 1985). Insulin inhibits the firing rate of hippocampal pyra midal neurons (Palovcik et aI., 1984) and of sympa thetic nerves (Sakaguchi and Bray, 1987) and stim ulates the maturation of cholinergic neurotransmis sion (Puro and Agardh, 1984). Streptozotocin (STZ) is established as a means of inducing experimental diabetes mellitus in rats when given by intraperitoneal administration (Junod et aI., 1968; Mordes and Rossini, 1981); this is achieved by way of inhibition of the insulin syn thesis in pancreatic islet cells (Bolaffi et aI., 1987; Zucker and Archer, 1988), decreased autophospho-
Received January 31, 1991; revised May 6, 1991; accepted May 23, 1991. Address correspondence and reprint requests to Prof. Dr. med. S. Hoyer at Department of Pathochemistry and General Neurochemistry, University of Heidelberg, 1m Neuenheimer Feld 220-221, D-6900 Heidelberg, Germany. Dr. A. Ding's present address is Mass Spectrometry Facility, University of California, San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0446, U.S.A. Abbreviations used: DA, dopamine; DOPAC, 3,4,-dihydroxy phenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; icv, intracerebroventricular; NA, noradrenaline; STZ, streptozotocin.
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rylation of the insulin receptor kinase (Kadowaki et aI., 1984), induced insulin resistance (Hussin and Skett, 1988), and also suppression of an insulin responsive glucose transporter (Garvey et aI., 1989). When applied to the brain, STZ may there fore be expected to interfere either with insulin syn thesis or with insulin receptor function or both. Provided brain insulin acts in the same manner as it does in nonnervous tissues (Kahn, 1985), an im pairment of the brain insulin/insulin receptor sys tem is thus likely to cause disturbances in cerebral glucose and related metabolism. Intracerebroven tricular (icv) administration of STZ in a subdiabe togenic dose did, in fact, result in decreased utili zation of glucose and increased release of lactate from the rat brain (Nitsch et aI., 1989), as well as in reduced concentrations of both ATP and creatine phosphate in rat brain cortex (Nitsch and Hoyer, 1991). The same treatment leads to an impairment of locomotor activity and of learning and memory in rats monitored by different psychometric tests (Mayer et aI., 1989, 1990). This may be due to ab normalities in the cholinergic system, in which nerve growth factor and choline acetyltransferase levels are altered in the septal region and the cortex subsequent to icv STZ administration, via de pressed cerebral glucose metabolism (Hellweg et aI., 1989). There are at least two different conditions In which cerebral glucose utilization is found to be preferentially perturbed in the presence of normo glycemia: aging (Gage et aI., 1984; Smith, 1984; Dastur, 1985; Hoyer, 1990) and incipient dementia of the Alzheimer type (DeLeon et aI., 1983; Hoyer et aI., 1988, 1991). The latter is characterized by an imbalance between (reduced) glucose utilization and (fairly normal or only slightly diminished) oxy gen consumption. There is evidence that the pertur bation in cerebral glucose metabolism precedes changes in glucose-related metabolic pathways (Hoyer, 1988; Hoyer and Nitsch, 1988), such as the metabolism of amino acids, which are assumed to be partly used to substitute for lacking glucose as a fuel for energy formation (Hoyer and Nitsch, 1989). ATP production (measured and calculated) was found to be reduced in the brain of patients suffer ing from incipient dementia of the Alzheimer type (Sims et aI., 1983; Hoyer, 1991). This energy deficit of �20% may obviously give rise to subsequent ab normalities in energy-dependent metabolic pro cesses (Siesjo, 1981; Erecinska and Silver, 1989), one of which is the storage and turnover of mono aminergic neurotransmitters (Tissari et aI., 1969; Mandel et aI., 1975). In brain affected by dementia of the Alzheimer type, alterations were found in the
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concentrations of the monoaminergic neurotrans mitters NA, dopamine (DA), and serotonin (5-HT), that of 5-HT being the most markedly reduced after acetylcholine (e.g., Gottfries et aI., 1983; Palmer et al., 1987, 1988; Reinikainen et al., 1988; Herregodts et aI., 1989). Therefore, the present study was designed to in vestigate whether or not the metabolism of the monoaminergic neurotransmitters NA, DA, and 5-HT changes subsequent to icv STZ injection and, if so, which neurotransmitters are involved and in which brain areas. It was also of interest to learn whether or not a rather general variation in mono aminergic neurotransmitters occurred or whether the latter changed more specifically. Here we demonstrate that NA drops markedly in the striatum and in the frontal and entorhinal corti ces; 5-HT drops most markedly in the entorhinal cortex with a concomitant increase in its turnover rate; and DA is unchanged in all brain areas inves tigated. Thus, a disturbance in cerebral glucose/ energy metabolism may precede abnormalities in distinct monoaminergic neurotransmitter concen trations in distinct brain areas.
MATERIALS AND METHODS Animals
One-year-old male Wistar rats weighing between 390 and 520 g were obtained from the Zentralinstitut fUr Ver suchstierzucht (Hannover, F. R.G.). They were housed two per cage with free access to water and food pellets (Altromin standard, no. 1320). An inverse day/night cycle ( 12 h/ 12 h) allowed the performance of experiments dur ing the active period of the circadian cycle. The animals were divided randomly into two groups for icv injection of either STZ (n 17) or artificial CSF (n 17). =
=
Surgical procedure
After anesthesia with chloral hydrate (240 mg/kg body wt i.p. in a 4% solution, Riedel-de Haen, Seelze, F. R. G.), each animal received a single unilateral (left side) icv in jection while held in a stereotaxic apparatus (Uhl, Asslar, F.R.G.). In the first group, STZ (Sigma, Munich, F. R. G. ) was injected in a subdiabetogenic dose ( 1. 5 mg/kg body wt). In the second group, which served as the control group, artificial CSF ( 120 mM NaCi, 3 mM KCl, 1. 15 mM CaCI2, 0.8 mM MgCi2, 27 mM NaHC03, and 0. 33 mM NaH2P04, pH adjusted to 7.2 by CO2 insufflation (all chemicals from Merck, Darmstadt, F. R.G.) was injected. The volume of fluid injected was the same in both groups (9 ILl). Three weeks after the icv injection, the animals of both groups were killed by freezing of their brains in situ with liquid nitrogen under steady-state conditions (anesthesia 0. 5 vol% halothane, nitrous oxide/oxygen 70:30) in arte rial normotension, normocapnia, normoxemia, normogly cemia, and normothermia and then stored at - 80°C until preparation of the different brain areas.
BRAIN MONOAMINE METABOLISM AFTER STREPTOZOTOCIN Sample preparation
Samples from the frontal cortex (43.2 ± 15.8 mg), the temporal cortex (48.0 ± 18.2 mg), the entorhinal cortex (36.9 ± 13. 1 mg), the anterior part of the hippocampus (47.1 ± 21.8 mg), and the dorsolateral striatum ( 1 1 1.2 ± 37.8 mg) were dissected from horizontal brain slices ( 1.5 mm thick) in a glove box maintained at - lYC. After weighing, the frozen brain samples were homogenized with a potter and sonicated for 1 min in 0.7 ml of an ice-cold HPLC buffer as described below. After centrif ugation at 50,000 g for 20 min and extraction with a 2-fold volume of n-heptane (Merck) to remove cell fragments, denatured proteins, and lipids, the aqueous phase was filtered through a OA5-f1m Millipore membrane filter, af ter which 10-30 f11 of each filtrate was injected directly into the chromatographic system, with exception of the filtrates from the striatum samples; these were diluted with a lO-fold volume of the HPLC buffer for measure ment of DA and 3 , 4-dihydroxyphenylacetic acid (DOPAC) levels. Chromatographic system
The HPLC system was made up of a Gilson 23 1140 1 autoinjector (Abimed, Langenfeld, F.R.G.), a Series 1 LC-pump (Perkin Elmer, Uberlingen, F.R.G.), a pulse damper (Biotronik, Maintal, F.R.G.), a precolumn, 20 x 4.6 mm, packed with Nucleosil NH2 5 m, an analytical column, 250 x 4.0 mm, packed with Nucleosil 100-5C18 (Macherey-Nagel, Duren, F.R.G.), and a Coulochem ESA detector (Environmental Sciences Assoc., Bedford, MA, U.S.A.) with a model 50 1 1 analytical cell. The de tector potentials were as follows: detector 1: + 0.32 V (oxidation); detector 2: -0040 V (reduction). The column temperature was controlled by means of a glass jacket coupled to a water thermostat (Colora, Lorch, F.R.G.) maintained at 28°C. The sampling and evaluation of data were carried out with Nelson 2600 chromatography soft ware, run on an IBM-compatible personal computer and a Nelson interface (ESWE, Sinsheim, F.R.G.). Separation of the biogenic amines NA, DA, DOPAC, homovanillic acid (HVA), 5-HT, and 5-hydroxyindole acetic acid (5-HIAA) was obtained with a 0. 1 M phos phate buffer (NaH2PO4 H20; Merck), pH 3. 1, contain ing 3 mM I-octane sulfonic acid Na salt (Janssen, Jeel, Belgium), 0.5 mM disodium ethylenediaminetetraacetate (Merck), 1.5 mM NaN3 (Ferak, Berlin, F.R.G.), and 24 vol% methanol (Merck) within 18 min. All chemicals were of analytical grade. Standards were obtained from Serva (Heidelberg, F.R.G.). •
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± 0.06 ng/mg wet wt in the entorhinal cortex, and 0. 34 ± 0.15 ng/mg wet wt in the striatum (Fig. O. STZ icv resulted in a significant decrease in the NA levels in the striatum (0.21 ± 0.06 ng/mg wet wt), in the entorhinal cortex (0.28 ± 0.05 ng/mg wet wt), and in the frontal cortex (0.38 ± 0. 04 ng/mg wet wt) (see Fig. 1). In the temporal cortex and the hippo campus, no differences from the control groups were observed.
5-HT tissue concentrations
The 5-HT levels in the frontal cortex (0.79 ± 0.17 ng/mg wet wt) and in the striatum (0.80 ± 0.22 ng/ mg wet wt) were about twice as high as in the other brain areas investigated (0.34 ± 0. 10 ng/mg wet wt in the hippocampus, 0.40 ± 0.14 ng/mg wet wt in the temporal cortex, and 0.43 ± 0.08 ng/mg wet wt in the entorhinal cortex in the control group) (Fig. 2). A significant difference between the icv STZ treated group and the control group was observed in the entorhinal cortex, where the 5-HT tissue con centration decreased to 0. 35 ng/mg wet wt (Fig. 2). 5-HIAA tissue concentration and 5-HT turnover rate
The 5-HIAA levels were highest in the striatum (0.86 ± 0.15 ng/mg wet wt) and in the frontal cortex (0.67 ± 0. 12 ng/mg wet wt) of the control group (Fig. 3). In the hippocampus (0.42 ± 0.09 ng/mg wet wt), the entorhinal cortex (0.37 ± 0.08 ng/mg wet wt), and the temporal cortex (0.28 ± 0.05 ng/mg wet wt), the 5-HIAA levels were lower, as were the 5-HT levels. STZ icv had no effect on the 5-HIAA tissue concentrations (Fig. 3). The 5-HT turnover rate, which was calculated as the 5-HIAA/5-HT ra tio, was found to be elevated from 0. 82 ± 0.26 (con trol) to 1. 21 ± 0.47 (STZ) in the entorhinal cortex (Fig. 4). No further changes were observed. 0.8 c::::J control c::::J STZ ICV 0.6 " �
Statistical analysis
Data are expressed as means ± SD (n 17). They were analyzed by means of the nonparametric two-tailed Mann-Whitney U test. Differences were accepted as sta tistically significant at p < 0.05. =
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RESULTS NA tissue concentrations
In the control group, NA levels were highest in the frontal cortex (0.44 ± 0.06 ng/mg wet wt), the mean values being lower in all other areas studied: 0.29 ± 0.06 ng/mg wet wt in the temporal cortex, 0.32 ± 0.08 ng/mg wet wt in the hippocampus, 0.34
CF
CT
CE
ST
HC
FIG. 1. Noradrenaline (NA) tissue concentrations (mean
±
SO, ng/mg wet wt) in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intracerebroventricu lar streptozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cortex; ST, striatum; HC, hippocampus. *p < 0.05 versus corresponding control group.
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'Aj 1.2
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FIG. 2. Serotonin (5-HT) tissue concentrations (mean
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±
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ng/mg wet wt) in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intracerebroventricular strep tozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cortex; ST, striatum; HC, hip pocampus. 'p < 0.05 versus corresponding control group.
Tissue concentrations of DA, DOPAC, and HVA; DA turnover rate
The DA levels in the different brain areas ranged from values near the detection limit in the temporal and entorhinal cortex (both 0.09 ± 0.02 ng/mg wet wt) and the hippocampus (0.06 ± 0.01 ng/mg wet wt), through moderate values in the frontal cortex (0.32 ± 0.09 ng/mg wet wt), to high values in the striatum (7.12 ± 1.11 ng/mg wet wt). The DA me tabolites were detected only in the striatum (DOPAC: 1.42 ± 0.34 ng/mg wet wt; HVA: 0.94 ± 0.20 ng/mg wet wt). No differences were found be tween the control group and the STZ group. The DA turnover rate in the striatum (0.29 ± 0.03), cal culated as DOPAC + HVA/DA, also did not differ significantly between these two groups. DISCUSSION
The results obtained in this study demonstrate an effect of icv STZ administration on the tissue con-
;; t � � ry E '"
o control EZ2I 5TZ icy
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FIG. 3. 5-Hydroxyindoleacetic acid (5-HIAA) tissue concen trations (mean ± SO, ng/mg wet wt) in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intra cerebroventricular streptozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cor tex; ST, striatum; HC, hippocampus. No statistically signifi cant differences between the groups. J Cereb Blood Flow Metab, Vol.12, No.1, 1992
CT
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FIG. 4. Serotonin (5-HT) turnover rate (mean
HC
SO) calcu lated as 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio in several rat brain areas. (Open bars), control group, n = 17; (hatched bars), intracerebroventricular streptozotocin (STZ icv) group, n = 17. CF, frontal cortex; CT, temporal cortex; CE, entorhinal cortex; ST, striatum; HC, hippocampus. 'p < 0.05 versus corresponding control group. ±
centrations of the monoaminergic neurotransmit ters NA and 5-HT in distinct brain areas. Under the experimental conditions studied, both the N A (-18%) and the 5-HT (-19%) concentrations were reduced to a similar extent in the entorhinal cortex. In the frontal cortex, only the NA tissue concentra tion was reduced, by 14%. Whereas these two neu rotransmitter systems are shown to be susceptible, the dopaminergic system does not seem to be in volved in these damaging mechanisms. This fact be came particularly obvious in the striatum, where the DA level was unaffected and the NA level was decreased by 38%. No changes were observed in the temporal cortex or in the hippocampus. The relationship between (pancreas) insulin effi cacy and brain neurotransmitters has been investi gated after the induction of experimental diabetes mellitus in rats in various studies. Th� most fre quent result reported has been a reduction in the cerebral tissue concentration of the 5-HT precursor tryptophan by 30--50% observed 1-4 weeks after intraperitoneal injection of STZ (Curzon and Fernando, 1977; Mackenzie and Trulson, 1978a; Trulson et aI., 1986). In contrast to these reports, according to which there were no alterations in the brain tissue levels of 5-HT or 5-HIAA, concentra tions of these were also reduced 2 weeks after in traperitoneal STZ treatment in the study of Kwok and Juorio (1987). In addition, a diminished synthe sis and turnover rate of cerebral 5-HT were found in rats with STZ-induced diabetes 2-3 weeks (Crandall et aI., 1981) and 4-6 weeks (Trulson et aI., 1986) after the injection. All the diabetes mellitus-related changes in brain referred to here were reported to be reversible after treatment with insulin. Besides 5-HT, the DA syn thesis in the striatum and the limbic forebrain was also found to be reduced (Trulson and Himmel,
BRAIN MONOAMINE METABOLISM AFTER STREPTOZOTOCIN 1985), as was the DA turnover rate in the striatum (Shimomura et aI., 1988). The turnover rate of NA correlated positively with the peripheral concentra tion of insulin (Smythe et aI., 1984; Trulson and Himmel, 1985). Thus, all monoaminergic neuro transmitter systems in the brain seem to be suscep tible to impairment during diabetes mellitus induced by intraperitoneal injection of STZ. Otherwise, when the immediate effect of insulin on the serotonergic metabolism of the brain was studied, icv administration of insulin was found to have no effect on the cerebral concentrations of tryptophan, 5-HT, or 5-HIAA (MacKenzie and Trulson, 1978a,b). When STZ was injected icv, Lackovic and Salkovic (1990) found an increase in the rat brain tissue levels of NA, DA, and 5-HT 1 week afterward. This result seems to be in contrast to our findings in this study. It must, however, be borne in mind that Lackovic and Salkovic (1990) pooled the whole brain for their studies and did not separate distinct brain areas from one another. Fur thermore, they used a different analytical tech nique, which was not clearly described in their ar ticle. In accordance with most of the aforemen tioned investigations, the data from this study show a relationship between insulin efficacy and mono aminergic neurotransmitter levels in the brain, in particular a reduction of 5-HT and NA in several brain areas. Assuming that insulin acts in the brain in the same way as in nonnervous tissues, our data suggest a direct influence of insulin on neuronal glu cose metabolism and thus on subsequent energy dependent events such as neurotransmitter metab olism in general (Erecinska and Silver, 1989) and storage/packing (Mandel et aI., 1975) and turnover/ reuptake (Tissari et aI., 1969) of neurotransmitters in particular. The specific pattern of the alterations may point to a selective vulnerability of the inner vated target cells. However, the cause of this re mains obscure. We speculate that the density of insulin receptors, which is known to be highest in the olfactory and limbic areas (Hill et aI., 1986; Werther et aI., 1987), may play a role in the inci dence of metabolic changes. The entorhinal cortex, which is a part of the limbic structures, is regarded as one of the most vulnerable brain areas as was demonstrated for dementia of the Alzheimer type (Hyman et aI., 1984). From a functional point of view, this area is of importance with respect to cog nitive processes: Entorhinal neurons carry afferent glutamatergic fibers, which provide the major cor tical input to the hippocampus via the perforant pathway (Hyman et aI., 1986, 1987). As detailed above, this study demonstrated disturbances in neurotransmitter metabolism in this brain area. The observed deficits in learning and memory
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(Mayer et aI., 1989, 1990) may also be at least partly related to the lesions of the serotonergic and nor adrenergic system. In particular, the serotonergic system is thought to be involved in cognitive pro cesses (Wenk et aI., 1987), whereas the noradren ergic system seems to be of minor significance in this respect (Gold and Welsh, 1987; Altman and Normile, 1988; Pontecorvo et aI., 1988). In conclu sion, we have demonstrated in this study that se lective losses in the tissue concentrations of the monoaminergic neurotransmitters NA and 5-HT may be due to an impairment of the cerebral glucose and energy metabolism after icv administration of STZ. Although it is not yet clear whether these al terations are the cause or the consequence of dam aging processes in some of their target regions, they are thought to be a metabolic basis of the deficits in learning and memory following intracerebral STZ application. Acknowledgment: We thank Roland Galmbacher, Gunter Galmbacher, Vera Apell, and Marina Traschutz for expert technical assistance and Jutta-Maria Herb, Janet Dodsworth, and Gunter Mayer for their helpful dis cussions of the manuscript. A. Ding was stipendiary of the Hirnliga e. V. Heidelberg (F.R. G. ).
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