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Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity
Brain Research 784 Ž1998. 305–315
Research report
Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity Makoto Bannai a , Masumi Ichikawa b , Masugi Nishihara a , Michio Takahashi a
a, )
Department of Veterinary Physiology, Veterinary Medical Science, The UniÕersity of Tokyo, Bunkyo-ku, Tokyo 113, Japan b Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Fuchu-shi, Tokyo, Japan Accepted 29 October 1997
Abstract In the ventromedial hypothalamus ŽVMH., g-aminobutyric acid ŽGABA. plays a role in regulating feeding and running behaviors. The GABA synthetic enzyme, glutamic acid decarboxylase ŽGAD., consists of two isozymes, GAD65 and GAD67 . In the present study, the phosphorothioated antisense oligodeoxynucleotides ŽODNs. of each GAD isozyme were injected bilaterally into the VMH of male rats, and food intake, body weight and locomotor activity were monitored. ODNs were incorporated in the water-absorbent polymer ŽWAP, 0.2 nmolrm l. so that ODNs were retained at the injection site. Each antisense ODN of GAD65 or GAD67 tended to reduce food intake on day 1 Žday of injections day 0. though not significantly. An injection combining both antisense ODNs significantly decreased food intake only on day 1, but body weight remained significantly lower than the control for 5 days. This suppression of body weight gain could be attributed to a significant increase in locomotor activity between days 3 and 5. Individual treatment with either ODNs did not change locomotor activity. The increase in daily locomotor activity in the group receiving the combined antisense ODNs occurred mainly during the light phase. Neither vehicle ŽWAP. nor control ODN affected food intake, body weight and locomotor activity. Histological studies indicated that antisense ODN distributed within 800 m m from the edge of the area where WAP was located 24 h after the injection gradually disappeared within days, but still remained within 300 m m distance even 7 days after the injection. Antisense ODN was effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias. Further, HPLC analysis revealed that antisense ODNs of GAD isozymes, either alone or combined, decreased the content of GABA by 50% in VMH 24 h after the injection. These results indicate that suppression of GABA synthesis by either of the GAD isozymes is synergistically involved in suppressing food intake and enhancing locomotor activity in rat VMH. q 1998 Elsevier Science B.V. Keywords: Antisense ODN; Ventromedial hypothalamus; Glutamic acid decarboxylase; GABA; Food intake; Locomotor activity
1. Introduction The ventromedial hypothalamus ŽVMH. has been shown to profoundly involved in regulating feeding w18,27,28x and sexual behavior w11,26,25x. In addition, we recently found that kainate-sensitive neurons in the VMH are involved in inducing hyper-running activity in rats w19,20x. These behavioral patterns are partially controlled by an inhibitory neurotransmitter, g-aminobutyric acid ŽGABA. w6,10,22,33x. The GABA synthetic enzyme, glutamic acid
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Corresponding author. Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, T o k y o 1 1 3 , Ja p a n . F a x : q 8 1 -3 -3 8 1 5 -4 2 6 6 ; E -m a il: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 4 9 - 8
decarboxylase ŽGAD; EC 4.1.1.15., consists of two isozymes, GAD65 and GAD67 , which derive from two distinctive genes and differ in molecular weight, neuronal localization and dependence on co-factors w5,7,8,13,14x. Functional differences between GAD65 and GAD67 in regulating sexual behavior were noticed when antisense oligodeoxynucleotide ŽODN. of respective GAD isozymes was administered into the medial hypothalamus of female rats w15x. Synthetic antisense ODNs complementary to specific mRNAs have been successfully used to block protein synthesis in a variety of cell culture systems w4,30x. More recently, intracerebral administration of antisense ODNs has been shown to modulate several behavioral functions w16,17,31x. However, the effectiveness of antisense ODNs is generally limited by a low efficiency in cellular uptake
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w12x and the lack of stability due to the degradation by nucleases w29x. Modification of the structure of ODNs by phosphorothioate formulations Ž S-oligos. has been shown to increase the resistance of ODNs to nucleases, though significant intracellular degradation still occurs via the lysosomal pathway w1x. Previously, we reported that the hydrogel of WAP is a useful carrier for slowly delivering neurotransmitters or their antagonists in the brain w19,20,32,33x. In the present study, antisense S-oligos to GAD65 and GAD67 were incorporated in the WAP gel and injected into rat VMH to investigate whether or how the blocking of each GAD isozyme synthesis by these treatments manifest changes in feeding behavior and locomotor activity.
2. Materials and methods 2.1. Animals and housing Adult male rats of the Wistar–Imamichi strain weighing 250–450 g were used in this study. All animals were housed under constantly controlled environmental conditions Žroom temperature, 23 " 18C; lights on from 0700 to 1900 h.. Illumination was supplied by fluorescent tubes. Pellet food ŽNihon-Nosan, MF Breeder, Japan. and water were available ad libitum. 2.2. Polymer–gel The water-absorbent polymer ŽWAP, Sanwet IM-1000, Sanyo-Kasei, Tokyo, Japan. which consists of co-bridged amylogen was used in this study. The water absorbing capacity of the polymer depends on the ionic strength of its environmental solution. In physiological saline, for example, the volume increases 80-fold and the polymer forms a hydro-polymer–gel, which is not affected by changes in environmental temperature and pressure. In this experiment, the maximum volume of physiological saline containing S-oligos Ž0.2 nmolrm l. was added to WAP to produce an 80-fold gel. 2.3. Oligodeoxynucleotides S-oligos and 5X biotinylated-S-oligos were obtained from Takara ŽShiga, Japan.. A 15-mer S-oligos complementary to the region spanning the translation start codon for each form of GAD was constructed. The sequence of the antisense S-oligos to GAD67 was 5X-TGC- CAT-CAG-CTCGGT-3X . The sequence of the antisense S-oligos to GAD65 was 5X-AGA-TGC- CAT-GGG-TTC-3X . The scrambled sequence for control S-oligos was 5X-CAG-GTG- CAT-ATCCCG-3X . Underlining indicates the sequence antisense to the translation start codon. Control S-oligos also includes the translation start codon. All of these sequences have no significant homology with known mRNA sequences ac-
cording to the Genbank database w24x. The secondary structure analysis using DNASIS program ŽHitachi Software Engineering, Tokyo. indicated that the supposed binding sites of antisense ODNs to GAD65 and GAD67 in corresponding mRNAs were not in tightly folded regions. Each S-oligo was incorporated into WAP at a concentration of 0.2 nmolrm l, and when antisense S-oligos to both GAD65 and GAD67 were simultaneously administered, 0.2 nmol of both antisense S-oligos were incorporated into 1 m l of WAP. 2.4. Statistics Data were analyzed using one-way factorial ANOVA. When there was a significant effect, Newman–Keuls analysis was used to test significance of post hoc effect. Since there was no significant difference between the data of WAP- and control ODN-injected groups in all experiments, the data of the two groups were combined for statistical analysis. 2.5. Experiment 1: behaÕioral study All surgical operations were performed between 1000 and 1600 h. The rats were anesthetized with pentobarbital sodium Ž45 mgrkg, i.p.. and maintained in a state of surgical anesthesia. In dwelling stainless steel cannula Ž0.9 mm, outer diameter. for hydro-polymer–gel, injection was stereotaxically implanted bilaterally into the VMH using the coordinates based on Paxinos and Watson w23x. The coordinates were 6.2 mm anterior from zero, 0.7 mm lateral to the sagittal sinus, and 9.3 mm below the endocranium. The cannula was fixed to the skull with anchor screws and dental cement. A sterile stainless steel stylet was inserted into the cannula to prevent the lumen from clot forming. After surgical operations, rats were transferred to individual cages equipped with an infra-red counter ŽMuromachi Kikai, Tokyo, Japan., and the locomotor activity of each rat was monitored. The output from the infra-red counters was transferred via an interface and recorded on the disks in a microcomputer Ž9801 VM, NEC, Tokyo, Japan. as a series of total counts per 30 min. Food intake and body weight were also monitored daily around 1100 h. The amount of food eaten a day was estimated by taking the difference of the weight of food in the container between 2 successive days. More than 5 days later, 1 m l of WAP containing S-oligoŽs. was injected into the VMH. A stainless steel injection cannula Ž0.5 mm, inner diameter. with polyethylene tubing was loaded with WAP containing the S-oligo. The loaded injection cannula was inserted into the indwelling guide cannula at about 1800 h, and the injection was done over a 30-s period under a free moving condition. WAP without S-oligos was also injected as another control. The injection cannula was then left in place for an
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Fig. 1. The effect of antisense ODNs on food intake. Day 0 was designated as the day of the injection of antisense ODNs. Values on each day are expressed as a ratio to the mean food intake for 5 days prior to the treatments. Each column and vertical bar represent the mean" S.E.M. The number of animals used is shown in parentheses. WAP, water-absorbent polymer Žvehicle.; AS, antisense ODN. ) P - 0.05 vs. pre-treatment value. †P - 0.05 vs. WAP q control ODN, GAD65 AS and GAD67 AS.
additional 2 min. The day of injection was designated as day 0, and locomotor activity, food intake and body weight were monitored until day 6. Locomotor activity on day Ž n. means that between 1900 h Žthe time of lights off. on day Ž n y 1. and 1900 h on day Ž n., and food intake on day Ž n. means that between 1100 h on day Ž n y 1. and 1100 h on day Ž n.. After the experimental period, the animals were anes-
thetized with pentobarbital sodium Ž45 mgrkg, i.p.., perfused with 200 ml of physiological saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline ŽPBS. at pH 7.3. The brain tissue was removed and stored in the same solution for 1 day at 48C and then in 30% sucrose in 0.1 M PBS overnight at 48C. Coronal sections were cut at 40 m m on a cryostat and stained with 0.1% cresyl violet ŽSigma, St. Louis, MO, USA.. Animals
Fig. 2. The effect of antisense ODNs on body weight. Day 0 was designated as the day of the injection of antisense ODNs. Values on each day are expressed as a ratio to the body weight on day 0. Each point and vertical bar represent the mean" S.E.M. The number of animals used is shown in parentheses. WAP, water-absorbent polymer Žvehicle.; AS, antisense ODN. ) P - 0.05 vs. WAP q control ODN, GAD65 AS and GAD 67 AS.
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Fig. 3. The effect of antisense ODNs on locomotor activity. Day 0 was designated as the day of the injection of antisense ODNs. The ratio to the mean locomotor activity for 5 days prior to the treatments is calculated in each treatment group, and values on each day are expressed as a difference between the ratio of antisense ODN treated-group and that of control groups ŽWAP q control ODN.. Each point and vertical bar represent the mean " S.E.M. The number of animals used is shown in parentheses. AS, antisense ODN. ) P - 0.05 vs. GAD65 AS andror GAD67 AS.
that had received treatments in sites other than VMH were excluded from the results. 2.6. Experiment 2: distribution of antisense ODN One microliter of WAP containing 0.2 nmol 5X biotinylated-antisense S-oligo to GAD67 was injected into bilateral
VMH through a stainless steel cannula under ether anesthesia. The cannula was left in place for an additional 2 min and then pulled out. After a period of 1, 3 and 7 days, the brain was removed and stored as described in Section 2.5. Coronal sections were cut at 40 m m on a cryostat, and free-floating sections were rinsed in 0.1 M PBS containing
Fig. 4. The effect of antisense ODNs on locomotor activity in the light Žleft panel. and dark Žright panel. phases on days 3–5. Day 0 was designated as the day of the injection of antisense ODNs. Values are expressed as a ratio of the mean locomotor activity for days 3–5 to that for 5 days prior to the treatments. Each column and vertical bar represent the mean " S.E.M. The number of animals used is shown in parentheses. WAP, water-absorbent polymer Žvehicle.; AS, antisense ODN. ) P - 0.05 vs. WAP q control ODN.
M. Bannai et al.r Brain Research 784 (1998) 305–315 Fig. 5. The distribution of antisense ODN labeled with FITC around the injection site. A, B, C and D: 24 h, 3 days, 5 days and 7 days after the injection, respectively. The dark area surrounded by fluorescence corresponds to the place where WAP Žan injection vehicle of water-absorbent polymer. had been deposited. Scale bar Žshown in A.: A, B, C, D, 300 m m. 309
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0.05% Triton-X, 1% BSA and 0.1% sodium azide ŽBSA– PBST. at pH 7.3 for 1 h at room temperature. The sections were then incubated with the first antibody against the specific markers for the neurons Žanti-MAP-2, Boehringer
Mannheim, Mannheim, 1:5000., for the astrocytes ŽantiGFAP, Dako, Glostrup, Netherlands, 1:2500., and for the microglias Žanti-monocytes, ED1, Biogenesis, Poole, USA, 1:5000. in PBST for 40 h at 48C. Following the reaction,
Fig. 6. The confocal microscopic images of antisense ODN containing-cells 3 days after the injection. ŽA. Antisense ODN labeled with FITC; ŽB. anti-MAP-2 immunoreactivity detected with rhodamine-labeled secondary antibody; ŽC. composite image of A and B; ŽD. and ŽE. composite images of antisense ODN labeled with FITC and anti-GFAP and anti-monocytes immunoreactivity detected with rhodamine-labeled secondary antibody, respectively. Green signal, antisense ODN; red signal, anti-MAP-2 for neurons ŽC., anti-GFAP for astrocytes ŽD., and anti-monocytes for microglias ŽE. immunoreactivity; yellow-orange signal, composite image of green and red signals. Scale bar Žshown in A.: A, B, C, D, E, 10 m m.
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Fig. 6 Žcontinued..
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the sections were washed three times with PBST. After washing, they were incubated with the second antibody Žanti-mouse IgG1, Southern Biotech, Birmingham, 1:50 for anti-MAP-2 and ED1; anti-rabbit Ig, Dako, 1:20 for antiGFAP. and streptavidinfluorescein ŽAmersham, Buckinghamshire, England. in PBST for 1 h at room temperature. Then the sections were washed five times with PBST. After the last wash, the sections were mounted on gelatin coated slides and covered with a cover glass using GelrMount ŽBiomeda, Foster City, CA, USA.. These slides were observed by fluorescent microscopy and confocal microscopy ŽCarl Zeiss, Oberkochen, Germany.. 2.7. Experiment 3: HPLC analysis for GABA One microliter of WAP containing S-oligoŽs. was injected into bilateral VMH under ether anesthesia. One day after the injection, the animals Ž6–8 animals for each group. were sacrificed by decapitation, and the brain was removed and coronally sliced by a tissue slicer Ž1 mm thick.. Only one section from each brain containing the VMH was selected and then the tissue surrounding a lump of WAP which had been deposited in the brain tissue was quickly punched out using a stainless steel pipe Ž1.1 mm inner diameter.. If the slicer cut a lump of WAP, the sample was discarded. The tissue samples Žfive for each. were homogenized in H 2 0rmethanol 1r1 Žabout 150 m l. and centrifuged for 20 min at 48C, 15,000 rpm. The supernatant Ž20 m l. was used for the assay of GABA and amino acids Žalanine, aspartate, glutamate, glutamine, glycine and taurine. by means of HPLC ŽEicom, Kyoto, Japan.. The protein concentration was measured by protein assay kit ŽBio-Rad, Hercules, USA., and tissue contents of GABA or other amino acids were normalized by protein contents.
3. Results 3.1. Experiment 1: behaÕioral study The effect of antisense ODNs on food intake is shown in Fig. 1 as a ratio to the mean food intake for 5 days prior to the treatments. Because no significant differences were observed between the vehicle ŽWAP. and the control ODN groups, the results of these two groups were combined as the control group in Fig. 1. Food intake on day 1 was significantly lower than that during pretreatment period in all the groups including the control group. Injection of combined antisense ODNs to both GAD isozymes significantly decreased food intake on day 1 as compared with the control group, and thereafter, the food intake recovered gradually to the pretreatment levels. Body weight on day 1 in the control group decreased slightly but not significantly when compared with that on day 0 ŽFig. 2.. This first day decrease in body weight was
significant and greatly amplified in the group being treated with both antisense ODNs. The body weight in this group remained suppressed for 5 days. Individual treatments with either one of the two antisense ODNs tended to shift the body growth curve 1 or 2 days behind that of the control group, though not significantly. Changes in daily locomotor activity following treatments with antisense ODNs are shown in Fig. 3. The ratio to the mean locomotor activity for 5 days prior to the treatments was calculated in each treatment group, and, since there was no significant difference between the groups treated with WAP and control ODN, they were combined as the control group. Values on each day are expressed as the difference between the ratio of the antisense ODN treated-group and that of the combined control group. A tendency to enhance a locomotor activity was observed in the combined ODNs treated group, but not in the individually treated groups. Thus, locomotor activity in the former group between days 3 and 5 was significantly higher than that in the latter groups. During this period Ždays 3–5., locomotor activity in the simultaneously treated group was significantly higher than that in the two control groups in the light phase, but not in the dark phase ŽFig. 4.. Treatments with antisense ODN to GAD65 or GAD67 did not affect daily total locomotor activity, but significantly reduced locomotor activity during the dark phase during days 3 and 5. 3.2. Experiment 2: distribution of antisense ODN Distribution of the antisense ODN signal around the injection site is shown in Fig. 5. Twenty-four hours after the injection, antisense ODN signal distributed within about 800 m m from the edge of area where WAP was located. Antisense ODN signal gradually disappeared with days, but still remained within a 300 m m distance even 7 days after the injection. Fig. 6 shows confocal pictures for antisense ODN signal ŽA., MAP-2 immunoreactivity ŽB., and composite image ŽC. 3 days after injection. Composite images for antisense ODN signal and anti-GFAP immunoreactivity ŽD., and anti-monocyte immunoreactivity ŽE. are also shown. In Fig. 6C,D and E, yellow-orange signal is composite image of green signal Žantisense ODN labeled with FITC. and red signal Žanti-MAP-2, anti-GFAP and antimonocytes immunoreactivity detected with rhodamine.. These images indicate that antisense ODN was effectively incorporated either in the neurons, astrocytes or microglias. 3.3. Experiment 3: HPLC analysis for GABA The contents of GABA and amino acids were measured in the VMH tissue thinly wrapping the injection site 24 h after the injection of antisense ODNs of GAD65 , GAD67 or both. The results are shown in Fig. 7 as a percent of those
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Fig. 7. The effect of antisense ODNs on GABA and amino acid contents in the VMH 24 h after the injection. Values are expressed as a percent of those of control groups ŽWAP q control ODN.. Each column and vertical bar represent the mean " S.E.M. The number of animals used is shown in parentheses. ) P - 0.05 vs. control groups.
of control groups ŽWAP q control ODN.. Antisense ODNs of GAD65 and GAD67 , either alone or combined, similarly reduced GABA content by about 50% within 24 h after the injection, while they did not alter contents of other amino acids.
4. Discussion The present study demonstrated that antisense S-oligos of GAD isozymes incorporated in WAP were efficiently taken up by cells including neurons around the injection site and exerted biological effects on feeding behavior and locomotor activity. It has been reported that, when S-oligos dissolved in saline were injected in mouse brain, they were detected in cells as early as 15 min after the injection, stable at least 8 h, and disappeared in 48 h w21x. In the present study, fluorescence labeling intensity was strongest 24 h after the injection of antisense ODN covering almost the entire VMH area and the signal still remained even 7 days after the injection. Further, immunohistochemical study suggested that neurons, as well as astrocytes and microglias, equally took up antisense ODN when examined on day 3. The reasons for these differences in distribution and cellular uptake of antisense ODNs may be that we used WAP as a carrier of antisense ODNs, which released them much more slowly into the surrounding tissue w32x. Antisense ODNs of GAD65 and GAD67 and combined antisense ODNs of them equally decreased GABA content in the VMH by about 50% in 24 h without affecting the content of other amino acids. This suggest that antisense
ODNŽs. used in the present study effectively blocked mRNA-dependent protein synthesis as expected leading to the reduction of GABA content in the VMH. The observation that both isozymes equally decreased GABA content suggest that, although they differ in intracellular localization and dependence on co-factors w7x, they are both similarly involved in maintaining GABA synthesis in GABAergic neurons in the VMH. Although the reason why combined antisense ODNs did not further reduced GABA content in the VMH is currently unclear, some compensatory mechanisms might take place to maintain GABA levels. Simultaneous administration of antisense ODNs to GAD65 and GAD67 significantly suppressed food intake on day 1 as compared with the control group. Each of the antisense ODNs, when administered independently, tended to suppress food intake in a similar way. It is well established that lesion of the VMH induces hyperphagia and obesity w3x, while stimulation of the VMH reduces food intake w2x, leading to a concept that the VMH is a satiety center. Involvement of GABAergic neurons in inhibiting neuronal activity in the VMH with a resultant increase in food intake is also suggested w9x. Taken together, the present results can be interpreted as follows: GABAergic neuronal activity permits feeding by reducing VMH neuronal activity, an effect compromised by GAD antisense ODNs. Although food intake recovered to pretreatment levels following day 2, body weight remained suppressed for 5 days after simultaneous administration of both antisense ODNs. This may be, at least partially, due to increased energy consumption by enhanced locomotor activity for
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days 3–5 by the treatment. This increase in locomotor activity originated mainly during the light phase, but not in the dark phase. We have previously shown that there are neurons inducing running in the VMH whose excitability is stimulated by kainate or a GABA antagonist, bicuculine and inhibited by GABA w33x. Further, chemical lesion of the VMH by high concentrations of kainate resulted in a decrease in locomotor activity during the dark phase Žour unpublished observation.. From these observations, we propose that GABA plays a role inhibiting the excitability of the neurons involved in inducing running in the VMH especially during the light phase when locomotor activity is almost totally suppressed in the rat, a nocturnal animal. This GABAergic inhibition on the VMH neurons may be released by antisense ODNs of GADs with a resultant increase in locomotor activity during the light phase. The present study provides no decisive information on the specific GAD molecule synthesizing GABA involved in regulating food intake and locomotor activity. Each GAD isozyme antisense ODN similarly reduced GABA content in the VMH by 24 h but did not significantly affect food intake and locomotor activity when administered individually. The effect of administration of combined antisense ODNs on the behavior, but not on GABA content, appeared to be additive. Based on the present results, it is probable that GAD65 and GAD67 play rather synergistic roles in regulating these VMH functions, though changes in behavior cannot be simply attributed to changes in GABA content. Simultaneous changes in GAD65 and GAD67 activity would be needed to affect feeding and locomotor activity. The reason for the discrepancy in the time course of antisense ODNs’ effects on feeding behavior and locomotor activity is currently unclear. Some compensatory mechanisms may occur for maintaining food intake at normal levels. Another possibility is the difference in the threshold for both phenotypes. In this concern, elucidation of changes in GABA content in the VMH throughout the experimental period following treatments with antisense ODNs to GADs, which was not done in the present study, would be helpful in resolving this problem. Further studies are needed to clarify the precise mechanisms of actions of antisense ODNs and specific roles of each of the GAD isozymes in regulating feeding behavior and locomotor activity.
Acknowledgements This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Culture and Sports, Japan, and ‘Research for the Future’ Program, The Japan Society for the Promotion of Science Ž97L00904..
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Research report
Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity Makoto Bannai a , Masumi Ichikawa b , Masugi Nishihara a , Michio Takahashi a
a, )
Department of Veterinary Physiology, Veterinary Medical Science, The UniÕersity of Tokyo, Bunkyo-ku, Tokyo 113, Japan b Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Fuchu-shi, Tokyo, Japan Accepted 29 October 1997
Abstract In the ventromedial hypothalamus ŽVMH., g-aminobutyric acid ŽGABA. plays a role in regulating feeding and running behaviors. The GABA synthetic enzyme, glutamic acid decarboxylase ŽGAD., consists of two isozymes, GAD65 and GAD67 . In the present study, the phosphorothioated antisense oligodeoxynucleotides ŽODNs. of each GAD isozyme were injected bilaterally into the VMH of male rats, and food intake, body weight and locomotor activity were monitored. ODNs were incorporated in the water-absorbent polymer ŽWAP, 0.2 nmolrm l. so that ODNs were retained at the injection site. Each antisense ODN of GAD65 or GAD67 tended to reduce food intake on day 1 Žday of injections day 0. though not significantly. An injection combining both antisense ODNs significantly decreased food intake only on day 1, but body weight remained significantly lower than the control for 5 days. This suppression of body weight gain could be attributed to a significant increase in locomotor activity between days 3 and 5. Individual treatment with either ODNs did not change locomotor activity. The increase in daily locomotor activity in the group receiving the combined antisense ODNs occurred mainly during the light phase. Neither vehicle ŽWAP. nor control ODN affected food intake, body weight and locomotor activity. Histological studies indicated that antisense ODN distributed within 800 m m from the edge of the area where WAP was located 24 h after the injection gradually disappeared within days, but still remained within 300 m m distance even 7 days after the injection. Antisense ODN was effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias. Further, HPLC analysis revealed that antisense ODNs of GAD isozymes, either alone or combined, decreased the content of GABA by 50% in VMH 24 h after the injection. These results indicate that suppression of GABA synthesis by either of the GAD isozymes is synergistically involved in suppressing food intake and enhancing locomotor activity in rat VMH. q 1998 Elsevier Science B.V. Keywords: Antisense ODN; Ventromedial hypothalamus; Glutamic acid decarboxylase; GABA; Food intake; Locomotor activity
1. Introduction The ventromedial hypothalamus ŽVMH. has been shown to profoundly involved in regulating feeding w18,27,28x and sexual behavior w11,26,25x. In addition, we recently found that kainate-sensitive neurons in the VMH are involved in inducing hyper-running activity in rats w19,20x. These behavioral patterns are partially controlled by an inhibitory neurotransmitter, g-aminobutyric acid ŽGABA. w6,10,22,33x. The GABA synthetic enzyme, glutamic acid
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Corresponding author. Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, T o k y o 1 1 3 , Ja p a n . F a x : q 8 1 -3 -3 8 1 5 -4 2 6 6 ; E -m a il: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 4 9 - 8
decarboxylase ŽGAD; EC 4.1.1.15., consists of two isozymes, GAD65 and GAD67 , which derive from two distinctive genes and differ in molecular weight, neuronal localization and dependence on co-factors w5,7,8,13,14x. Functional differences between GAD65 and GAD67 in regulating sexual behavior were noticed when antisense oligodeoxynucleotide ŽODN. of respective GAD isozymes was administered into the medial hypothalamus of female rats w15x. Synthetic antisense ODNs complementary to specific mRNAs have been successfully used to block protein synthesis in a variety of cell culture systems w4,30x. More recently, intracerebral administration of antisense ODNs has been shown to modulate several behavioral functions w16,17,31x. However, the effectiveness of antisense ODNs is generally limited by a low efficiency in cellular uptake
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w12x and the lack of stability due to the degradation by nucleases w29x. Modification of the structure of ODNs by phosphorothioate formulations Ž S-oligos. has been shown to increase the resistance of ODNs to nucleases, though significant intracellular degradation still occurs via the lysosomal pathway w1x. Previously, we reported that the hydrogel of WAP is a useful carrier for slowly delivering neurotransmitters or their antagonists in the brain w19,20,32,33x. In the present study, antisense S-oligos to GAD65 and GAD67 were incorporated in the WAP gel and injected into rat VMH to investigate whether or how the blocking of each GAD isozyme synthesis by these treatments manifest changes in feeding behavior and locomotor activity.
2. Materials and methods 2.1. Animals and housing Adult male rats of the Wistar–Imamichi strain weighing 250–450 g were used in this study. All animals were housed under constantly controlled environmental conditions Žroom temperature, 23 " 18C; lights on from 0700 to 1900 h.. Illumination was supplied by fluorescent tubes. Pellet food ŽNihon-Nosan, MF Breeder, Japan. and water were available ad libitum. 2.2. Polymer–gel The water-absorbent polymer ŽWAP, Sanwet IM-1000, Sanyo-Kasei, Tokyo, Japan. which consists of co-bridged amylogen was used in this study. The water absorbing capacity of the polymer depends on the ionic strength of its environmental solution. In physiological saline, for example, the volume increases 80-fold and the polymer forms a hydro-polymer–gel, which is not affected by changes in environmental temperature and pressure. In this experiment, the maximum volume of physiological saline containing S-oligos Ž0.2 nmolrm l. was added to WAP to produce an 80-fold gel. 2.3. Oligodeoxynucleotides S-oligos and 5X biotinylated-S-oligos were obtained from Takara ŽShiga, Japan.. A 15-mer S-oligos complementary to the region spanning the translation start codon for each form of GAD was constructed. The sequence of the antisense S-oligos to GAD67 was 5X-TGC- CAT-CAG-CTCGGT-3X . The sequence of the antisense S-oligos to GAD65 was 5X-AGA-TGC- CAT-GGG-TTC-3X . The scrambled sequence for control S-oligos was 5X-CAG-GTG- CAT-ATCCCG-3X . Underlining indicates the sequence antisense to the translation start codon. Control S-oligos also includes the translation start codon. All of these sequences have no significant homology with known mRNA sequences ac-
cording to the Genbank database w24x. The secondary structure analysis using DNASIS program ŽHitachi Software Engineering, Tokyo. indicated that the supposed binding sites of antisense ODNs to GAD65 and GAD67 in corresponding mRNAs were not in tightly folded regions. Each S-oligo was incorporated into WAP at a concentration of 0.2 nmolrm l, and when antisense S-oligos to both GAD65 and GAD67 were simultaneously administered, 0.2 nmol of both antisense S-oligos were incorporated into 1 m l of WAP. 2.4. Statistics Data were analyzed using one-way factorial ANOVA. When there was a significant effect, Newman–Keuls analysis was used to test significance of post hoc effect. Since there was no significant difference between the data of WAP- and control ODN-injected groups in all experiments, the data of the two groups were combined for statistical analysis. 2.5. Experiment 1: behaÕioral study All surgical operations were performed between 1000 and 1600 h. The rats were anesthetized with pentobarbital sodium Ž45 mgrkg, i.p.. and maintained in a state of surgical anesthesia. In dwelling stainless steel cannula Ž0.9 mm, outer diameter. for hydro-polymer–gel, injection was stereotaxically implanted bilaterally into the VMH using the coordinates based on Paxinos and Watson w23x. The coordinates were 6.2 mm anterior from zero, 0.7 mm lateral to the sagittal sinus, and 9.3 mm below the endocranium. The cannula was fixed to the skull with anchor screws and dental cement. A sterile stainless steel stylet was inserted into the cannula to prevent the lumen from clot forming. After surgical operations, rats were transferred to individual cages equipped with an infra-red counter ŽMuromachi Kikai, Tokyo, Japan., and the locomotor activity of each rat was monitored. The output from the infra-red counters was transferred via an interface and recorded on the disks in a microcomputer Ž9801 VM, NEC, Tokyo, Japan. as a series of total counts per 30 min. Food intake and body weight were also monitored daily around 1100 h. The amount of food eaten a day was estimated by taking the difference of the weight of food in the container between 2 successive days. More than 5 days later, 1 m l of WAP containing S-oligoŽs. was injected into the VMH. A stainless steel injection cannula Ž0.5 mm, inner diameter. with polyethylene tubing was loaded with WAP containing the S-oligo. The loaded injection cannula was inserted into the indwelling guide cannula at about 1800 h, and the injection was done over a 30-s period under a free moving condition. WAP without S-oligos was also injected as another control. The injection cannula was then left in place for an
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Fig. 1. The effect of antisense ODNs on food intake. Day 0 was designated as the day of the injection of antisense ODNs. Values on each day are expressed as a ratio to the mean food intake for 5 days prior to the treatments. Each column and vertical bar represent the mean" S.E.M. The number of animals used is shown in parentheses. WAP, water-absorbent polymer Žvehicle.; AS, antisense ODN. ) P - 0.05 vs. pre-treatment value. †P - 0.05 vs. WAP q control ODN, GAD65 AS and GAD67 AS.
additional 2 min. The day of injection was designated as day 0, and locomotor activity, food intake and body weight were monitored until day 6. Locomotor activity on day Ž n. means that between 1900 h Žthe time of lights off. on day Ž n y 1. and 1900 h on day Ž n., and food intake on day Ž n. means that between 1100 h on day Ž n y 1. and 1100 h on day Ž n.. After the experimental period, the animals were anes-
thetized with pentobarbital sodium Ž45 mgrkg, i.p.., perfused with 200 ml of physiological saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline ŽPBS. at pH 7.3. The brain tissue was removed and stored in the same solution for 1 day at 48C and then in 30% sucrose in 0.1 M PBS overnight at 48C. Coronal sections were cut at 40 m m on a cryostat and stained with 0.1% cresyl violet ŽSigma, St. Louis, MO, USA.. Animals
Fig. 2. The effect of antisense ODNs on body weight. Day 0 was designated as the day of the injection of antisense ODNs. Values on each day are expressed as a ratio to the body weight on day 0. Each point and vertical bar represent the mean" S.E.M. The number of animals used is shown in parentheses. WAP, water-absorbent polymer Žvehicle.; AS, antisense ODN. ) P - 0.05 vs. WAP q control ODN, GAD65 AS and GAD 67 AS.
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Fig. 3. The effect of antisense ODNs on locomotor activity. Day 0 was designated as the day of the injection of antisense ODNs. The ratio to the mean locomotor activity for 5 days prior to the treatments is calculated in each treatment group, and values on each day are expressed as a difference between the ratio of antisense ODN treated-group and that of control groups ŽWAP q control ODN.. Each point and vertical bar represent the mean " S.E.M. The number of animals used is shown in parentheses. AS, antisense ODN. ) P - 0.05 vs. GAD65 AS andror GAD67 AS.
that had received treatments in sites other than VMH were excluded from the results. 2.6. Experiment 2: distribution of antisense ODN One microliter of WAP containing 0.2 nmol 5X biotinylated-antisense S-oligo to GAD67 was injected into bilateral
VMH through a stainless steel cannula under ether anesthesia. The cannula was left in place for an additional 2 min and then pulled out. After a period of 1, 3 and 7 days, the brain was removed and stored as described in Section 2.5. Coronal sections were cut at 40 m m on a cryostat, and free-floating sections were rinsed in 0.1 M PBS containing
Fig. 4. The effect of antisense ODNs on locomotor activity in the light Žleft panel. and dark Žright panel. phases on days 3–5. Day 0 was designated as the day of the injection of antisense ODNs. Values are expressed as a ratio of the mean locomotor activity for days 3–5 to that for 5 days prior to the treatments. Each column and vertical bar represent the mean " S.E.M. The number of animals used is shown in parentheses. WAP, water-absorbent polymer Žvehicle.; AS, antisense ODN. ) P - 0.05 vs. WAP q control ODN.
M. Bannai et al.r Brain Research 784 (1998) 305–315 Fig. 5. The distribution of antisense ODN labeled with FITC around the injection site. A, B, C and D: 24 h, 3 days, 5 days and 7 days after the injection, respectively. The dark area surrounded by fluorescence corresponds to the place where WAP Žan injection vehicle of water-absorbent polymer. had been deposited. Scale bar Žshown in A.: A, B, C, D, 300 m m. 309
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0.05% Triton-X, 1% BSA and 0.1% sodium azide ŽBSA– PBST. at pH 7.3 for 1 h at room temperature. The sections were then incubated with the first antibody against the specific markers for the neurons Žanti-MAP-2, Boehringer
Mannheim, Mannheim, 1:5000., for the astrocytes ŽantiGFAP, Dako, Glostrup, Netherlands, 1:2500., and for the microglias Žanti-monocytes, ED1, Biogenesis, Poole, USA, 1:5000. in PBST for 40 h at 48C. Following the reaction,
Fig. 6. The confocal microscopic images of antisense ODN containing-cells 3 days after the injection. ŽA. Antisense ODN labeled with FITC; ŽB. anti-MAP-2 immunoreactivity detected with rhodamine-labeled secondary antibody; ŽC. composite image of A and B; ŽD. and ŽE. composite images of antisense ODN labeled with FITC and anti-GFAP and anti-monocytes immunoreactivity detected with rhodamine-labeled secondary antibody, respectively. Green signal, antisense ODN; red signal, anti-MAP-2 for neurons ŽC., anti-GFAP for astrocytes ŽD., and anti-monocytes for microglias ŽE. immunoreactivity; yellow-orange signal, composite image of green and red signals. Scale bar Žshown in A.: A, B, C, D, E, 10 m m.
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Fig. 6 Žcontinued..
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the sections were washed three times with PBST. After washing, they were incubated with the second antibody Žanti-mouse IgG1, Southern Biotech, Birmingham, 1:50 for anti-MAP-2 and ED1; anti-rabbit Ig, Dako, 1:20 for antiGFAP. and streptavidinfluorescein ŽAmersham, Buckinghamshire, England. in PBST for 1 h at room temperature. Then the sections were washed five times with PBST. After the last wash, the sections were mounted on gelatin coated slides and covered with a cover glass using GelrMount ŽBiomeda, Foster City, CA, USA.. These slides were observed by fluorescent microscopy and confocal microscopy ŽCarl Zeiss, Oberkochen, Germany.. 2.7. Experiment 3: HPLC analysis for GABA One microliter of WAP containing S-oligoŽs. was injected into bilateral VMH under ether anesthesia. One day after the injection, the animals Ž6–8 animals for each group. were sacrificed by decapitation, and the brain was removed and coronally sliced by a tissue slicer Ž1 mm thick.. Only one section from each brain containing the VMH was selected and then the tissue surrounding a lump of WAP which had been deposited in the brain tissue was quickly punched out using a stainless steel pipe Ž1.1 mm inner diameter.. If the slicer cut a lump of WAP, the sample was discarded. The tissue samples Žfive for each. were homogenized in H 2 0rmethanol 1r1 Žabout 150 m l. and centrifuged for 20 min at 48C, 15,000 rpm. The supernatant Ž20 m l. was used for the assay of GABA and amino acids Žalanine, aspartate, glutamate, glutamine, glycine and taurine. by means of HPLC ŽEicom, Kyoto, Japan.. The protein concentration was measured by protein assay kit ŽBio-Rad, Hercules, USA., and tissue contents of GABA or other amino acids were normalized by protein contents.
3. Results 3.1. Experiment 1: behaÕioral study The effect of antisense ODNs on food intake is shown in Fig. 1 as a ratio to the mean food intake for 5 days prior to the treatments. Because no significant differences were observed between the vehicle ŽWAP. and the control ODN groups, the results of these two groups were combined as the control group in Fig. 1. Food intake on day 1 was significantly lower than that during pretreatment period in all the groups including the control group. Injection of combined antisense ODNs to both GAD isozymes significantly decreased food intake on day 1 as compared with the control group, and thereafter, the food intake recovered gradually to the pretreatment levels. Body weight on day 1 in the control group decreased slightly but not significantly when compared with that on day 0 ŽFig. 2.. This first day decrease in body weight was
significant and greatly amplified in the group being treated with both antisense ODNs. The body weight in this group remained suppressed for 5 days. Individual treatments with either one of the two antisense ODNs tended to shift the body growth curve 1 or 2 days behind that of the control group, though not significantly. Changes in daily locomotor activity following treatments with antisense ODNs are shown in Fig. 3. The ratio to the mean locomotor activity for 5 days prior to the treatments was calculated in each treatment group, and, since there was no significant difference between the groups treated with WAP and control ODN, they were combined as the control group. Values on each day are expressed as the difference between the ratio of the antisense ODN treated-group and that of the combined control group. A tendency to enhance a locomotor activity was observed in the combined ODNs treated group, but not in the individually treated groups. Thus, locomotor activity in the former group between days 3 and 5 was significantly higher than that in the latter groups. During this period Ždays 3–5., locomotor activity in the simultaneously treated group was significantly higher than that in the two control groups in the light phase, but not in the dark phase ŽFig. 4.. Treatments with antisense ODN to GAD65 or GAD67 did not affect daily total locomotor activity, but significantly reduced locomotor activity during the dark phase during days 3 and 5. 3.2. Experiment 2: distribution of antisense ODN Distribution of the antisense ODN signal around the injection site is shown in Fig. 5. Twenty-four hours after the injection, antisense ODN signal distributed within about 800 m m from the edge of area where WAP was located. Antisense ODN signal gradually disappeared with days, but still remained within a 300 m m distance even 7 days after the injection. Fig. 6 shows confocal pictures for antisense ODN signal ŽA., MAP-2 immunoreactivity ŽB., and composite image ŽC. 3 days after injection. Composite images for antisense ODN signal and anti-GFAP immunoreactivity ŽD., and anti-monocyte immunoreactivity ŽE. are also shown. In Fig. 6C,D and E, yellow-orange signal is composite image of green signal Žantisense ODN labeled with FITC. and red signal Žanti-MAP-2, anti-GFAP and antimonocytes immunoreactivity detected with rhodamine.. These images indicate that antisense ODN was effectively incorporated either in the neurons, astrocytes or microglias. 3.3. Experiment 3: HPLC analysis for GABA The contents of GABA and amino acids were measured in the VMH tissue thinly wrapping the injection site 24 h after the injection of antisense ODNs of GAD65 , GAD67 or both. The results are shown in Fig. 7 as a percent of those
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Fig. 7. The effect of antisense ODNs on GABA and amino acid contents in the VMH 24 h after the injection. Values are expressed as a percent of those of control groups ŽWAP q control ODN.. Each column and vertical bar represent the mean " S.E.M. The number of animals used is shown in parentheses. ) P - 0.05 vs. control groups.
of control groups ŽWAP q control ODN.. Antisense ODNs of GAD65 and GAD67 , either alone or combined, similarly reduced GABA content by about 50% within 24 h after the injection, while they did not alter contents of other amino acids.
4. Discussion The present study demonstrated that antisense S-oligos of GAD isozymes incorporated in WAP were efficiently taken up by cells including neurons around the injection site and exerted biological effects on feeding behavior and locomotor activity. It has been reported that, when S-oligos dissolved in saline were injected in mouse brain, they were detected in cells as early as 15 min after the injection, stable at least 8 h, and disappeared in 48 h w21x. In the present study, fluorescence labeling intensity was strongest 24 h after the injection of antisense ODN covering almost the entire VMH area and the signal still remained even 7 days after the injection. Further, immunohistochemical study suggested that neurons, as well as astrocytes and microglias, equally took up antisense ODN when examined on day 3. The reasons for these differences in distribution and cellular uptake of antisense ODNs may be that we used WAP as a carrier of antisense ODNs, which released them much more slowly into the surrounding tissue w32x. Antisense ODNs of GAD65 and GAD67 and combined antisense ODNs of them equally decreased GABA content in the VMH by about 50% in 24 h without affecting the content of other amino acids. This suggest that antisense
ODNŽs. used in the present study effectively blocked mRNA-dependent protein synthesis as expected leading to the reduction of GABA content in the VMH. The observation that both isozymes equally decreased GABA content suggest that, although they differ in intracellular localization and dependence on co-factors w7x, they are both similarly involved in maintaining GABA synthesis in GABAergic neurons in the VMH. Although the reason why combined antisense ODNs did not further reduced GABA content in the VMH is currently unclear, some compensatory mechanisms might take place to maintain GABA levels. Simultaneous administration of antisense ODNs to GAD65 and GAD67 significantly suppressed food intake on day 1 as compared with the control group. Each of the antisense ODNs, when administered independently, tended to suppress food intake in a similar way. It is well established that lesion of the VMH induces hyperphagia and obesity w3x, while stimulation of the VMH reduces food intake w2x, leading to a concept that the VMH is a satiety center. Involvement of GABAergic neurons in inhibiting neuronal activity in the VMH with a resultant increase in food intake is also suggested w9x. Taken together, the present results can be interpreted as follows: GABAergic neuronal activity permits feeding by reducing VMH neuronal activity, an effect compromised by GAD antisense ODNs. Although food intake recovered to pretreatment levels following day 2, body weight remained suppressed for 5 days after simultaneous administration of both antisense ODNs. This may be, at least partially, due to increased energy consumption by enhanced locomotor activity for
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days 3–5 by the treatment. This increase in locomotor activity originated mainly during the light phase, but not in the dark phase. We have previously shown that there are neurons inducing running in the VMH whose excitability is stimulated by kainate or a GABA antagonist, bicuculine and inhibited by GABA w33x. Further, chemical lesion of the VMH by high concentrations of kainate resulted in a decrease in locomotor activity during the dark phase Žour unpublished observation.. From these observations, we propose that GABA plays a role inhibiting the excitability of the neurons involved in inducing running in the VMH especially during the light phase when locomotor activity is almost totally suppressed in the rat, a nocturnal animal. This GABAergic inhibition on the VMH neurons may be released by antisense ODNs of GADs with a resultant increase in locomotor activity during the light phase. The present study provides no decisive information on the specific GAD molecule synthesizing GABA involved in regulating food intake and locomotor activity. Each GAD isozyme antisense ODN similarly reduced GABA content in the VMH by 24 h but did not significantly affect food intake and locomotor activity when administered individually. The effect of administration of combined antisense ODNs on the behavior, but not on GABA content, appeared to be additive. Based on the present results, it is probable that GAD65 and GAD67 play rather synergistic roles in regulating these VMH functions, though changes in behavior cannot be simply attributed to changes in GABA content. Simultaneous changes in GAD65 and GAD67 activity would be needed to affect feeding and locomotor activity. The reason for the discrepancy in the time course of antisense ODNs’ effects on feeding behavior and locomotor activity is currently unclear. Some compensatory mechanisms may occur for maintaining food intake at normal levels. Another possibility is the difference in the threshold for both phenotypes. In this concern, elucidation of changes in GABA content in the VMH throughout the experimental period following treatments with antisense ODNs to GADs, which was not done in the present study, would be helpful in resolving this problem. Further studies are needed to clarify the precise mechanisms of actions of antisense ODNs and specific roles of each of the GAD isozymes in regulating feeding behavior and locomotor activity.
Acknowledgements This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Culture and Sports, Japan, and ‘Research for the Future’ Program, The Japan Society for the Promotion of Science Ž97L00904..
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