Material and methods them remained for 2 h in a state of quiet wakefulness

P . Torterolo et al. Brain Research 884 2000 68 –76 69 This suppression of cellular discharge was reversed follow- maintained with a gas mixture of halothane 1–3 in ing the iontophoretic application of GABA antagonists oxygen. Thereafter, the head was positioned in a stereo- A into the DRN during AS [25]. In addition, microdialysis taxic frame and the calvarium was exposed. Stainless steel experiments have demonstrated that GABA release in the screw electrodes were placed in the frontal and parietal DRN increases during AS, and that microperfusion of the bones for recording the electroencephalogram EEG and GABA antagonist picrotoxin decreases the time spent in into the orbital portion of the frontal bone to record eye AS while GABA agonist muscimol increases the duration movements EOG. Deep bipolar electrodes were im- of this state [37]. These facts suggest that GABAergic planted in both lateral geniculate nuclei in order to monitor inhibition may be responsible for the cessation of activity ponto-geniculo-occipital waves PGO. A Winchester plug in serotonergic neurons that accompanies this state. How- connected to these electrodes and a chronic head-restrain- ever, the origin of the GABAergic control of DRN neurons ing device were bonded to the calvarium with acrylic during AS is not clear. cement. A small hole, 5 mm in diameter, which was made We and others have used the expression of c-fos during in the calvarium overlying the cerebellar cortex, was then AS induced by microinjection of carbachol into the NPO filled with bone-wax. This hole was subsequently used to of the cat to examine neural activity during this state provide access for drug administration. At the end of these  [9,33,34,48,62–64]. The AS-carbachol state resembles surgical procedures, an analgesic Buprenex , 0.01 mg naturally occurring AS polygraphically, electrophysiologi- kg, i.m. was administered. Incision margins were kept cally and behaviorally [3,12,32,55]. Furthermore, in a clean and a topical antibiotic was administered on a daily similar model, carbachol microinjection into the NPO was basis. After the animals had recovered from these surgical found to produce a cessation of firing of serotonergic procedures, they were adapted to the head-restraining neurons in the DRN, which is similar to that which occurs device for 2 weeks. during natural AS [50]. Using the AS-carbachol model, we previously demonstrated that the DRN has a larger number 2.3. Experimental procedures of c-fos-expressing neurons during AS-carbachol than during wakefulness and that the majority of these neurons In the experimental animals, carbachol 0.8 mg in 0.2 ml are not serotonergic [63]. of saline was microinjected into the NPO AP 22 to 23, In the present study, using a double-labeling procedure L 1 to 2, H 23.5 to 25, according to Berman’s atlas [6] for GABA and Fos, we found that the number of activated using a 2-ml Hamilton syringe. After the animals had spent GABAergic neurons in the DRN increases during AS- approximately 2 h in the AS-carbachol state, they were carbachol. We therefore hypothesize that these cells may be deeply anesthetized with sodium pentobarbital i.p. 60 GABAergic neurons that inhibit serotonergic neurons of mg kg and perfused for immunocytochemistry see the DRN during this state. Preliminary results of this study below. Within this time period, the c-fos protein product have been presented in abstract form [53]. Fos is known to reach optimal concentration [9,34,62]. The control animals underwent the same surgical and habituation procedures as the experimental animals. All of

2. Material and methods them remained for 2 h in a state of quiet wakefulness

before they were euthanized. In one control cat C4, 2.1. Animals saline 0.2 ml was microinjected into the NPO 2 h before the animal was euthanized. The EEG, EOG, PGO and neck Seven male adult cats three experimental and four electromyogram EMG, obtained using acutely placed controls were used in this study. The animals were bipolar electrodes were recorded and stored in a Power  obtained from and determined to be in good health by the Macintosh G3 microcomputer using SUPERSCOPE software. UCLA Division of Laboratory Animal Medicine. All of the experimental procedures were conducted in accord 2.4. Histological procedures with the Guide for the Care and Use of Laboratory Animals 7th edition, National Academy Press, Washington The animals were perfused under deep anesthesia with 1 DC, 1996 and approved by the Chancellor’s Animal l of heparinized saline followed by 1.5 l of a solution of Research Committee of the UCLA Office for Protection of 4 paraformaldehyde, 15 saturated picric acid and 0.5 Research Subjects. of glutaraldehyde in phosphate buffer PB, 0.1 M, pH 7.4. Subsequently, perfusion with 500 ml of the same solution 2.2. Surgical procedures with 10 sucrose was carried out. The brainstem was removed and immersed in a postfixative solution for 24 h; Before anesthesia, the animals were premedicated with the solution consisted of 2 paraformaldehyde, 15  Xylazine 2.2 mg kg, i.m., atropine 0.04 mg kg, i.m. saturated picric acid and 10 of sucrose in PB. Following   and antibiotics Cefazolin and Bicillin , i.m.. Anesthesia postfixation, the tissue was kept for 3 days in a solution of was first induced with ketamine 15 mg kg, i.m. and sucrose 25 and sodium azide 0.1 in PB. Thereafter 70 P the brainstem was frozen and cut into 14 mm-thick sections out in seven sections for each animal one section every using a Reichert–Jung cryostat. The sections were stored 500 mm. The mean number of immunostained neurons in a solution of 0.1 sodium azide, 0.25 in phosphate Fos1, GABA1 Fos1 and GABA2 Fos1 on each side buffered saline PBS, 0.1 M. of the brain and for each animal were then calculated. Brainstem sections were first immunostained for Fos. These means were used to compare stained neurons during For this purpose, free-floating sections from selected brain AS-carbachol and wakefulness n53 and n54, respective- stem sections were incubated overnight in a rabbit poly- ly. clonal Fos antiserum Fos Ab5; Oncogene Research Prod- The serotonergic area extends beyond the cytoarchitec- ucts Calbiochem, La Jolla, CA, USA at a dilution of tonic limits of the DRN [42,60]. In order to standardize the 1:40 000 in PBS. The sections were rinsed four times in area of analysis, the lateral borders were defined by a PBS for a total duration of 30 min and then incubated for parasagittal plane traced from the lateral border of the 90 min in a biotinylated donkey anti-rabbit immuno- main fascicle of the medial longitudinal fascicle mlf, see globulin G 1:700. Subsequently, the sections were incu- Fig. 3B. The dorsal and ventral borders of the DRN were bated with the ABC complex Vector ABC Elite kit, determined according to standard criteria [52]. Serotoner- 1:500 for 60 min. After rinsing again, the tissue was gic neurons were observed throughout the defined area of reacted for 10–20 min with 0.6 nickel ammonium analysis. In caudal sections, special care was taken to sulfate, 0.02 diaminobenzidine tetrahydrochloride avoid including neurons of the dorsal tegmental nucleus of Sigma and 0.015 hydrogen peroxide in 50 ml of 50 Gudden in this analysis. mM tris buffer, pH 7.5. Photomicrographs were obtained using a Spot digital In order to identify GABAergic neurons, polyclonal camera attached to an Olympus B360 microscope. Images  guinea pig antibodies raised against GABA-keyhole limpet were analyzed using Adobe PHOTOSHOP software with a hemocyanin conjugated with glutaraldehyde NT-108, Power Macintosh G3 computer. Conventional and Protos were employed. After the sections were treated for Nomarsky optics were employed. A 1003 oil immersion Fos immunostaining, they were incubated overnight with objective lens was used to measure the diameter of soma GABA antibody 1:3500 and normal donkey serum profiles. The distribution of labeled neurons was deter- NDS; 3. Then, after the sections were rinsed, they were mined from drawings using a camera lucida attachment. incubated for 60 min with biotinylated donkey anti-guinea The level of statistical significance of the difference pig antibody 1:300 plus NDS. After another rinse, the between the mean number of immunoreactive neurons in tissue was incubated in the ABC complex 1:200 for 60 control and AS-carbachol cats was evaluated using the min. Finally, the tissue was exposed to diaminobenzidine Student’s t-test. The criterion chosen to discard the null without nickel enhancement. hypothesis was P,0.05. Serotonin immunocytochemistry was performed on brainstem sections of both an AS-carbachol cat and an awake cat which were also both employed for GABA and

3. Results

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