Bilateral lesion of the nucleus basalis with ibotenic acid infusions in young and aged rats results in the degeneration of cholinergic neurons which innervate the ...
Neurochemical Research, VoL 17, No. 4, 1992, pp. 345-350
High-Affinity Transport of Choline and Amino Acid Neurotransmitters in Synaptosomes from Brain Regions after Lesioning the Nucleus Basalis Magnocellularis of Young and Aged Rats J. Gomeza 1, C. Arag6n 1, and C. Gim6ne# ,2 (Accepted October 1, 1991)
Bilateral lesion of the nucleus basalis with ibotenic acid infusions in young and aged rats results in the degeneration of cholinergic neurons which innervate the cortex. As expected, high-affinity uptake of choline was decreased in the frontal cortex subsequent to the lesion. Twenty one days after surgery there was a significantly decrease of the transport rate of GABA, glutamate and glycine in the frontal cortex of young rats, but those activities showed a recovery six months after lesion. On the contrary, 12-month old rats lesioned with the same experimental protocol showed no recovery of the transport rates in the frontal cortex. Uptake of choline, GABA, glutamate and glycine has also been studied in other areas of the brain, namely, hippocampus, olfactory bulb and cerebellum. The present results suggest that lesioning the nucleus basalis of rats led to a more effective and permanent impairment of some biochemical functions of the brain, when compared to young lesioned animals, and also suggest a functional relationship between the nucleus basalis and other areas of the brain. KEY WORDS: Nucleus basalis lesions; amino acid neurotransmitters; transport.
INTRODUCTION
cholinergic neurons which constitute most part of its neuronal population (5,6). The nucleus basalis magnocellularis in rats is homologous to the nucleus basalis of Meynert in primates and project its neurons to the cerebral cortex (7-9). The destruction of these cholinergic neurons by electrocoagulation or by injection of the excitotoxin ibotenic acid (10,11) produces a significant decrease in a c e t y l c h o l i n e p r o d u c t i o n and choline acetyltransferase (E.C. 2.3.1.6) activity in the ipsilateral neocortex similar to those seen in patients with Alzheimer's disease (12.13). Amino acids as glutamate and y-aminobutyric acid (GABA) are becoming considered as the major transmitters in the cerebral cortex and more recently it has been shown they are affected in some types of dementias (14-15). High-affinity sodium-dependent uptake of amino
On the past few years, the importance of CNS cholinergic neurons in learning and memory is supported by a consistent body of biochemical and pharmacological evidence (1). Impairments in the basal forebrain cholinergic system found both during normal aging and in Alzheimer's disease have led to the cholinergic hypothesis in order to explain geriatric memory dysfunctions (2-4). Neurotoxic lesions of the nucleus basalis magnocellularis in the rat bring about the degeneration of the I Departamento de Biologfa Molecular, Centro de Biologfa Molecular. Facultad de Ciencias, Universidad Aut6noma de Madrid, Spain. z Address reprint requests to: Prof. C. Gim6nez, Centro de Biologfa Molecular, Facultad de Ciencias, Universidad Aut6noma de Madrid, 28049-Madrid, Spain.
345 0364-3190/92/0400-0345506.50/09 1992PlenumPublishingCorporation
346 acid neurotransmitters by neural tissue is thought to represent re-uptake by nerve endings and to be a major means of termination of their actions (16). Whether aging or bilateral lesions have different cortical neurochemical effects related to the capacity of neurotransmitter recapture by neurons or glial cells is unknown at the present time. The purpose of the present study was to induce a cholinergic hypofunction in young and aged rats after lesioning the nucleus basalis magnoceltularis and to study the effects on the sodium-dependent transport of choline and some amino acid neurotransmitters in discrete areas of the brain. Our results show that transport of choline, GABA, glutamate and glycine in the cortex and other related areas is significantly reduced after cholinergic lesions.
EXPERIMENTAL PROCEDURE Chemicals. [2,3-3H]GABA (25Ci/mmol), [methyl-3H]choline chloride (80 Ci/mmol) and [2-3H]glycine (49Ci/mmol) were obtained from New England Nuclear, Boston, MA, U.S.A. [G-3H]glutamate (56Ci/mmol) was from Amersham International. All other chemicals and solvents were of the analytical or reagent grade and were used without further purification. All aqueous solutions were prepared with destilled deionised water and filtered through Millipore filters (0.45 ~m). GeneralProtoeoL Three- and twelve-months old male Wistar rats were anesthesied with 50mg/kg of sodium pentobarbital (IP) and then placed in a stereotaxic apparatus. Bilateral lesions of the nucleus basalis were made by stereotaxic injection of ibotenic acid 25 nmol in 1.0 bd of 50 mM sodium phosphate buffer (pH 7.4) with a Hamilton syringe. The injection lasted 10 rain and the syringe was left in place for 5 min after the completion of the infusion. The following coordinates were used: 0.9 mm posterior to bregma, 2.8 mm lateral, 6.8 mm below dura (17). Control rats received sham surgery but no infusion into the nucleus basalis. At the end of the experiments the placement and size of the lesions were checked by histological examination. At the times stated in the Results the rats were killed by decapitation, and their brains were rapidly removed and dissected for histology and neurochemical measurements according to the method of Glowinsky and Iversen (18). Preparation of Synaptosomes. Synaptosomes were prepared essentially as previously described by Booth and Clarck (19). After dissections of the brain samples were rapidly removed and homogenized (1:10) in ice-cold isolation medium (0.33 M sucrose, 1 mM K-EDTA, 10 mM HEPES-Tris, pH 7.4) in a Dounce-type glass homogenizer. The homogenate was centrifuged (1,300g, 3min), the pellet discarded, and the supernatant centrifuged at 17,000 g for 10 min. The pellet was resuspended (1:2) in isolation medium and layered on the top of a discontinuous Ficoll gradient consisting of 3 ml of 7.5% (wt/vol) Ficoll in isolation medium on 3.5 ml of 13% (wtPr Ficoll in isolation medium and centrifuged at 98,000 g for 30 rain. The synaptosomal fraction was then siphoned off from the 7.5/13% Ficoll interphase, diluted 10-fold with isolation medium, and centrifuged (15,000 g, 10
Gomeza, Arag6n, and Gim~nez min). The pellets were washed once within a maximum of 3 h after preparation. Transport Studies. Transport was measured as previously described (20). Unless otherwise stated, 20 tzl portions of the synaptosomal suspension (equivalent to 0.1 mg of protein) were preincubated for 5 min at 37~ in modified Krebs-Henseleit medium (128 mM NaC1, 5 mM KC1, 2.7 mM CaC12, 1.3 mM MgSO4, 10 mM glucose, 20 mM HEPES-Tris, pH 7.4). The uptake was started by addition of 80 tzl of a solution kept at 37~ and containing the desired ionic composition together with the labelled amino acid. After incubation with gentle agitation, uptake was terminated by diluting the incubation mixture with 3 ml of modified Krebs-Henseleit medium kept at room temperature and then immediately filtering through a moistened MiUipore filter (HAWP 02500, 0.45 ~m pore size) attached to a vacuum assembly. Filters were rinsed once with another 3 ml of modified KrebsHenseMt medium. The dilution, filtration, and washing procedures were performed within 15 s. Filters were dried, placed in microvials, and their radioactivity measured by scintillation spectrometry. Results were corrected for a control obtained by diluting the synaptosomal suspension before adding the radioactive substrate solution. All transport media were filtered through Millipore filters (0.45 ~m) before the experiments to avoid possible bacterial contamination. The osmolarity of all solutions was kept constant during the uptake experiments. Protein Determination. Syrtaptosomal protein content was determined according to the method of Lowry et al. (21). Statistical Analysis of the Data. Results are expressed as mean _+SEM, unless otherwise stated. Assessment of statistically significant differences between values was done by two-tailed Student's t test for non-paired samples.
RESULTS Survival was greatest than 90% either in the 3-month or 12-month injured rats group. One week after surgery, ibotenic acid injected animals had lost aprox. 15% of the average initial weight in the 3-month animals group and aprox. 11% in the 12-month animals group. At the end of the experiments the animals looked healthy and they did not differ significantly from sham operated in weight. Figure 1 shows a time course of the high-affinity choline transport in synaptosomes from frontal cortex of 3- and 12-month rats. The synaptosomes reached an equilibrium 5-7 min after incubation and 30 s was chosen as the right time to perform further transport experiments. The Figure 1 also shows that synaptosomes from old rats have aprox. 20% less capacity for transporting choline than those from younger animals. An experiment was conducted in order to follow the baseline levels of the high-affinity uptake of choline, GABA, glutamate and glycine after lesion. The Figure 2 shows the results of transport rates measured at 3, 7, 14, and 21 days, and six months after a bilateral lesion in three-month old rats. The transport capacity of syn-
Brain Damage and Neurotransmitter Uptake
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was indistinguishable from that of the sham operated rats as far as uptake of glutamate and GABA is concerned, whereas there was a slight decrease in the transport of choline at the same time.
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Figure 3 shows the effect of a bilateral lesion in 12month old rats on the high-affinity transport of choline in frontal cortex, hippocampus, cerebellum and olfactory bulb six months after lesion. Uptake of choline in frontal cortex decreased over 40%, aprox. 18% and 20% in hippocampus and olfactory bulb respectively whereas no significantly changes were found in cerebellum when compared with sham operated animals at the same age. The results in Figure 4 shows the effect of the lesion on the uptake of GABA in the former mentioned areas from 18-months old rats. As shown in the Figure 4, the lesion caused a decrease in the GABA uptake in frontal cortex (35%) and olfactory bulb (20%) and no changes in hippocampus and cerebellum. Glutamate transport is reduced by 30% and 15% in frontal cortex and olfactory bulb respectively after lesion, with no significant changes in hippocampus and cerebellum (Figure 5), and high-affinity transport of glycine is affected significantly by 20% and 35% in frontal~
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Fig. 2. Effects of bilateral nucleus basalis lesions on 3-month old rats frontal cortex uptake of choline, GABA, glutamate and glycine. Tansport rates were assayed at the specified intervals after receiving bilateral infusions of ibotenic acid in their nucleus basalis. Synaptosomes (Img of protein/ml) were preincubated for 5 rain and then labeled choline (0.2 IxM) (O), GABA (0.1 ~M) (O), glutamate (0.1~M) (F-q) or glycine (21xM) (ll), was added in Krebs-Henseleit medium and the mixture left incubating for 30 s. 100% of uptake corresponds to 0.52 • 0.02 pmol of choline/mg of protein; to 5.93 4- 0.21 pmol of GABA/mg of protein; to 12.61 • 0.91 pmol of L-glutamate/mg of protein, and to 16.20 - 1.00 pmol of glycine/mg of protein. Results are expressed as means • SEM of separate experiments.
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aptosomes from frontal cortex follow similar patterns for the different neurotransmitters studied although with different inhibition extent. T w e n t y one days after lesion, a 35% decrease in high-affinity choline uptake was found in frontal cortex, whereas the transport of glutamate and G A B A showed aprox. 20% o f decrease respectively. Six months after surgery the performance of the lesioned rats
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Fig. 3. High-affinity choline uptake in different brain regions of 18month old rats after bilateral ibotenic acid lesions of the nucleus basalis. Choline uptake was assayed six months after lesion. Synaptosomes (ling of protein/ml)were preincubated for 5 min and then labeled choline (0.2 p,M) was added in Krebs-HenseMt medium for 30 s. Results are expressed as mean • SEM of five separate experiments. *p
