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Monoaminergic uptake in synaptosomes prepared from frozen brain tissue samples of normal and narcoleptic canines
Bra& Research, 588 (1992) 115-119 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
115
BRES 17982
Monoaminergic uptake in synaptosomes prepared from frozen brain tissue samples of normal and narcoleptic canines D e l p h i n e V a l t i e r , W i l l i a m C. D e m e n t a n d E m m a n u e l M i g n o t Stanford Unicersity, School of Medicine, Sleep Disorders Center, Palo Alto, CA 94304 (USA) (Accepted 17 March 1992)
Key words: Synaptosome; Frozen tissue; Noradrenaline; Dopamine; Serotonin uptake; Narcolepsy; REM sleep
Canine narcolepsy, a model of the human disorder, is associated with altered catecholamine but not serotonin (5-HT) metabolism in some brain areas, particularly the amygdala. A possible explanation for these global changes could be the existence of specific defects in monoamine uptake processes. We have studied the uptake of ['~Hlnorepinephrine (NE), [3H]dopamine (DA) and [3H]5-HT in synaptosomes prepared from cortex and amygdala of narcoleptic and control Doberman pinscher brains. Since narcoleptic canines are relatively few in number, we have used a specific brain freezing procedure that has been reported to allow restoration of metabolically functional tissue upon thawing. Preliminary studies comparing monoamine uptake in fresh and frozen brain samples of both groups of dogs were carried out and demonstrated that this procedure significantly altered serotoninergic but not noradrenergic and dopaminergic uptake. All further investigations were then done on synaptosomes prepared from frozen samples. Our results demonstrate that synaptosomal uptake of [3HINE, [3H]DA and [3H]5-HT in cortex and amygdala are not altered in narcolepsy.
INTRODUCTION
Narcolepsy is a genetically determined REM sleep disorder of unknown origin affecting humans and animals 3'4. At Stanford University, we have established a colony of Doberman pinschers that transmit the disease as a fully penetrant autosomal recessive trait. This model has been used extensively to determine the neurochemical basis of narcolepsy and the most consistently found abnormality reported (two independently replicated studies) has been an increased concentration of DOPAC and dopamine (DA) in the amygdala6'13'~5. In this study, we hypothesized that a possible explanation for this modification could be altered DA uptake in this brain region. In order to test this hypothesis, we have measured monoamine uptake in synaptosomes prepared from frozen control and narcoleptic brain structures. Synaptosomes are artificially prepared nerve endings that are able to accumulate neurotransmitters against a concentration gradient ~. Monoamine uptake that can be mea-
sured in synaptosomes is a specific, energy dependent process which is sensitive to metabolic inhibitors, temperature, ion concentration and various neuropharmacological compounds 7. It is therefore a suitable model that has been used by numerous investigators. Metabolically viable synaptosomes usually need to be prepared from fresh tissue since cellular respiration and structural organization of nerve endings need to be preserved. In this study, we have used a method that allows us to prepare active synaptosomes from tissue that has been slowly frozen and rapidly thawed 4'5's. This technique has been used successfully on human and animal tissue to study monoamine uptake ~7 and it allowed us to gather age-matched control and narcoleptic dog brains. Our results indicate that this technique can be successfully used to prepare active synaptosomes from frozen dog brains. We also showed that monoamine uptake of DA, norepinephrine (NE) and serotonin (5-HT) is not altered in cortex and amygdala of the narcoleptic dog.
Correspondence: E. Mignot, Stanford University, School of Medicine, Sleep Disorders Center, 701 Welch Road, Suite 2226, Paio Alto, CA 94304, USA.
116 MATERIALS A N D M E T H O D S
Subj¢cl$ Twelve Doberman pinschers of the Stanford Canine Narcolepsy Colony were included in the study. Animals were individually housed in the Division of Laboratory Animal Medicine of Stanford University. All experiments were performed in accord with the current edition of the NIH guide for the care and use of laboratory animals. Six animals were narcoleptic (born from two narcoleptic parents: 2 females and 4 males, aged 21.83 +_7.10 months) and had shown clear, spontaneous cataplectic and drug-induced attacks during the Food Elicited Cataplexy Test (FECT). The 6 other animals were control dogs (born from two normal parents: 2 females and 4 males, aged 23.66___4.25 months). Narcoleptic and control animals were born from 4 and 3 different crosses respectively in order to avoid litter effects in the comparison. Dogs were drug free for at least 2 weeks at the time of the sacrifice,
Tissue collection and storage procedure One control and one affected animal (roughly age-matched) were sacrificed the same day using an i.e. overdose of thiopental (60 mg/kg). Brains were quickly removed and the amygdala and frontoparietai cortex anatomically dissected at room temperaturel2. Brain pieces were weighed, individually placed in eppendorf tubes and frozen according to Hardy et al.'~. Tubes were collected in a plastic bag, wrapped in cotton wool, placed at -20°C for 8-9 h, then transferred to -70°C. A thermistance probe was inserted in one of the brain pieces to record local tissue temperature during the freezing procedure. When tissue had reached -70°C, samples were placed in a normal Revco box until use. On the day of the experiment, frozen tissue was rapidly thawed by immersion of the tube in a 37°C waterbath for 15-20 s (per 100 mg of tissue) and transferred immediately to an ice-cold 0.32 M sucrose, 5 mM Na-HEPES (N-2hydroxethyl piperazine-N-2-ethanesulfonic acid) pH 4 buffer for synaptosomal preparation.
Synaptosomal preparation Brain pieces were homogenized at 0°C using a poter teflon-glass homogenizer (12 strokes, 800 rpm, clearance 0.5 mm) and then crude homogenate centrifuged (Beckman L8 ultracentrifuge, fixed angel rotor Ti 70.1) at 5,000 rpm (w:t = 1.57.107) at 4°C. The resulting supernatant fraction was layered onto half of its volume of 1.2 M sucrose solution and centrifuged at 50,(100 rpm (w:t = 1.610~) to yield at the sucrose interface a crude fraction that contained synaptosomes, myelin and microsomes, This fraction was carefully collected, diluted in ice-cold 0.32 M sucrose, 5 mM HEPES solution, layered onto half of its volume of (1.8 M sucrose solution and centrifuged again under the same conditions (50,000 rpm, w2t = 1.610s) to generate a pellet enriched in synaptosomes. The pellet was then resuspended in 1 ml of 0.32 M sucrose, 5 mM HEPES/100 mg of original tissue to reach a concentration of 0.4-0.5 mg of proteins/ml and kept on ice for no longer than 3 h. Protein concentration was determined according to the Bradford technique:.
Synaptosomal [ 3Hlneurotransmitter uptake Optimum assay conditions for monoamine uptake were determined by studying the time-course of [3H]NE accumulation at different protein concentrations, 0.1-0.7 mg protein/ml. Accumulation of ['~H]NE into purified synaptosome preparation was linear during the initial 5 min of the incubation at 37°C. The initial uptake also shows a linear relationship with the protein concentrations (synaptosome concentrations) in the range of 0.1-0.5 mg protein/ml. Therefore, in the subsequent studies an incubation time of 5 rain at a final synaptosome concentration of 0.4-0.5 mg protein/ml was employed.
K M (the apparent affinity constant of the transporter) and Vmax (the maximum velocity of the transporter system) were determined for each experiment. Aliquots of the synaptosome-enriched homogenates were incubated in a buffer (pH 7.4) containing (in mM): NaCi 140, KCi 5, MgCI 2 I, glucose 10, HEPES 10 with ascorbic acid 0.5, disodium EGTA 0.1 and pargyline 0.05 Ca monoamine oxydase inhibitor). Fifty /.tl of homogenate was added at 0°C to 400 p,i of the incubation buffer. After equilibrating at 37°C for 10 min, 50 ~l of [3H]neurotransmitter was added to yield a final concentration ranging from 0.002 to 0.5 gM ([3H]NE, [3H]DA, [3H]5-HT). The synaptosomal suspensions were then incubated an additional 5 min to determine monoamine uptake. Non-specific uptakes were determined at 0°C. For [3H]DA and ['~H]5-HT, filters were pretreated with polyethyleneimine (0.1%) for 3-5 min before filtration. To stop the incubation, 5 ml of ice-cold uptake buffer was added in each tube and synaptosomes were separated by filtration on G F / C glass fiber filters (Whatman) with moderate vacuum, then filters were washed twice with 5 ml of ice-cold uptake buffer to eliminate unincorporated [3H]neurotransmitter. Filters were dissolved in scintillation mixture (Cytoscint) for liquid scintillation counting in a Beckman counter (Model LS 3801).
Electron microscopy in order to assess the quality of the synaptosomal preparations, all samples were examined using electron microscopy. Synaptosomes were incubated at 37°C for 30 min and pelleted in a bench centrifuge at room temperature (10,000 rpm for 2 rain). The pellet was then fixed by immersion in phosphate buffer (0.1 M pH 7.24) containing glutaraldehyde (2%) and paraformaldehyde (!%) overnight at 0°C and postfixed the following day in 1% osmium tetroxide. Samples were then embedded in a plastic mixture (VCD-HXSA) I~ consisting of vinylcyclohexylene silicone 200 (0.5 ml) and dimethyl-amino ethanol (0.5 ml). Sections were cut using a LKB ultramicrotome and viewed on a Philips 410 electron microsope. Pictures of diverse magnifications were taken to assess the purity of the synaptosomal fraction and the integrity of synaptosomal structures.
Lactate dehydrogenase assay The synaptosome concentration in the preparation was also evaluated by determining lactate dehydrogenase (LDH) activity. LDH is a cytoplasmic marker ~, exclusively present in synaptosomes but not mitochondria or other contaminating fragments. Total synaptosome lactate dehydrogenase activity was assayed using the method of. Johnson I°. LDH activity was expressed in international units per ml (amount of LDH that causes a decrease of 0.001 per minute in the absorbance at 340 nm of the 3 ml reaction mixture in a I cm curet).
Data analysis and statistics K M and Vmax of the uptake transporter were estimated using a linear regression after Lineweaver-Burk transformation, Values reported are means :t:S.E.M. of independent kinetic experiments. Statistical analysis was performed using SYSTAT, Inc. (Evanston, IL) on a Macintosh SE.
Drugs The following substances were used: [ethyl-l-3H(N)dopamine (spec. act. 37 Ci/mmol; L[7-3H]noradrenaline (spec. act. 14.3 Ci/mmol) and 5-hydroxy['~H]tryptamine creatinine sulphate (spec. act. 21 Ci/mmol) NEN Boston, MA. Ascorbic acid, pargyline, disodium EDTA (ethylenglycoi-bis-aminoethylether-N,N'-tetracetic acid) and LDH diagnostic kit NADH were purchased from Sigma (St Louis, MO).
Fig. 1. Electron microscopy photographs of synaptosomes prepared from slowly frozen/fast thawed amygdala samples of control (A) and narcoleptic (B) dog brains. Bar = 4 ~m.
lC
!ill
118 TABLE !
TABLE !I
Comparison of ['~H]NE, [~H]DA and ['~H]5-HT uptake in s),naptosomes prepared from fresh and slowly frozen/fiast thawed cortical samples
Comparison of I SH]NE, [ 3H]DA and [ 3H]5-HT uptake in synaptosomes prepared from slowly frozen ~fast thawed cortical samples from narcoleptic (N) and control (C) dogs
K M values are given in 10 -7 M, I/max values in pmol/mg protein/rain. Values are means + S.E.M. of 6 samples for [3H]NE uptake and of 3-4 samples for [3H]DA and [3H]5-HT uptake. No slatisticai differences were observed using a Student's t-test except for ['aH]5-HT uptake (Vmax, P < 0.001).
K M values are given in 10 -7 M, Vmax values in p m o l / m g protein/ min. Values are means +-S.E.M. of 6 samples for [3H]NE uptake and samples for [3H]DA and [3H]5-HT uptake. No statistical differences were observed using a Student's t-test or a non-parametric Mann-Whitney U-test.
['~H]NE
[ 3H]DA
13H]NE
['~H]5-HT
I';H]DA
[ 3H]5-HT
KM
V,,, ,
KM
V,.....
K M
V,....
KM
V,.a.~
KM
V,,,~x
KM
V,,,.,
Fresh
2.82 + 1.07
0.775 +0.158
2.76 + 2.16
I. ! 63 +0.575
0.539 +_0.05
1.917 +_0.095
C
0.580 + 0.156
0.713 + 0.353
0.756 + 0.282
4.706 + 3.00
1.96 + 0.982
1.144 + 0.573
Frozen
2.39 +_0.74
0.656 +0.162
1.69 0.731 1.08 +_0.62 +_0.202 +_0.27
0.776 +_0.065
N
0.961 +0.127
0.577 +0.226
0.912 +0.530
3.40 + 1.67
1.13 +-0.83
0.956 +-0.884
RESULTS
Effects of freezing and thawing on the synaptosomal preparation Using electron microscopy, no morphological differences were found between synaptosomes prepared from fresh or frozen control and narcoleptic dog cortex brain samples (data not shown). Synaptosomal fractions (membrane-bound vesicle containing structures) were reasonably homogenous and uncontaminated by mitochondria (laminar apperance) or myelin and broken membranes (less than 50% contamination). Synaptosome sizes (diameters) ranged between 0.6 and 0.9 ~m. Electron microscopic analysis of the slowly frozen/fast thawed amygdala preparations did not show differences between narcoleptic and control dog brains. LDH activity was also determined in all the synaptosomal samples tested. No significant differences were found between values obtained in fresh or frozen samples (control or narcoleptic, cortex or amygdala).
Effects of freezing and thawing on synaptosomal uptake of monoamines The effect of the freezing/thawing procedure was explored using the 3 monoamines. Results are presented in Table I. A tendency to a decreased Vmax was observed in frozen samples for all monoamines but was statistically significant only for serotonin uptake. No significant changes in KM were observed when fresh or slowly frozen/fast thawed canine cortex tissue was t,sed. All subsequent experiments were carried out using slowly frozen/fast thawed tissues.
Morphological differences in synaptosomes obtained from control and narcoleptic animals Protein and LDH concentrations in amygdala preparations did not differ between narcoleptic and
control samples. For controls and narcoleptics values were respectively: 0.438 + 0.064 mg protein/ml, 0.481 + 0.050 mg protein/ml (Student's t-test, P > 0.297) and 1077.5 + 208.6 LDH units; 1032.5 + 175.4 LDH units (Student's t-test, P > 0.464). In both groups, a significant correlation between protein concentrations and LDH units was observed, thus indicating that the same proportion of synaptosomes were present in both preparations (Controls, r = 0.790 and Narcoleptics, r = 0.903). Electron microscopy performed on the samples also confirmed the first results observed in the cortical preparations. No major alterations were found in either narcoleptic or control dog tissues after slow freezing and rapid thawing. Fig. 1A, B represents electron microscopy photographs of synaptosomes prepared from the amygdala of control and narcoleptic dogs respectively.
Investigation of uptake parameters in narcolepsy Uptake of [aH]NE, [aH]DA and [aH]5-HT was assessed in synaptosomes prepared from cortex and amygdala. Uptake parameters for the 3 radioligands TABLE III
Comparison of 13H]NE, [';H]DA and 13H]5.HT uptake in synaptosprees prepared from slowly frozen/fast thawed amygdala samples from narcoleptic and control dogs. K M values are given in 10 -7 M, Vmax values in p m o l / m g protein/ rain. Values are means +_S.E.M. of 6 samples for [3H]NE and [aH]DA uptake and 3 samples for [3H]5-HT uptake. No statistical differences were observed using a Student's t-test or a non-parametric MannWhitney U-test.
[SHINE
['*H]DA
l SHI5-HT
KM
V,,ax
KM
V,mx
C
2.15 +_0.66
0.397 +_0.105
1.81 +_0.34
0.509 1.76 +_0.082 +_0.26
KM
1.55 +_0.47
N
1.50 +_0.37
0.409 +_0.124
1.00 5:0.307
0.482 +-0.128
1.32 +_0.25
2.13 _+0.85
E, ax
119
were investigated on the same tissue preparation, each experiment consisting of a simultaneous analysis of a control and a narcoleptic dog sample. No statistical differences were found between narcoleptic and control dogs for the kinetic uptake parameters obtained for the three neurotransmitters in cortex or amygdala (Tables II and Ill). DISCUSSION
One of the most consistent neurochemical abnormalities reported in the brain of narcoleptic canines is a change in DA content and metabolism in the amygdala 6a3'~5. In this study, we have investigated the uptake of monoamines in synaptosomes prepared from the amygdala of narcoleptic and control dogs. No difference was observed, suggesting that the neurochemical changes reported in previous studies are not secondary to abnormal uptake processes in this structure. In addition, electron microscopy on the samples did not reveal any gross morphological abnormalities in the synaptosomal vesicular formation. An interesting aspect of this study is the use of slowly frozen/rapidly thawed tissue 9, instead of fresh tissue to assess synaptosomal function. This technique is particularly useful if tissue samples are scarce since it affords the possibility of performing several independent studies on the same brain. This technique has been used in rodents where the brain could be processed immediately after death and in humans where variable lag between death and processing always has to be taken into account in the interpretation of the result, in this study, we demonstrated that this technique is also practical for another large-brain-sized mammal, the dog. Using this species, our protocol demonstrated only a very limited difference in monoamine uptake and morphology between fresh and frozen preparations. In conclusion, this study demonstrates the usefulness of the slow freezing/rapid thawing technique to prepare viable synaptosomal preparations in species where resources are scarce. Using this technique, we could not observe any changes in monoamine uptake parameters in the amygdala of the narcoleptic dog.
Acknowledgements. This work was supported by Grant NIH NS 23724 to W.C.D. We thank Fran Thomas for her participation in the electron microscopy studies; Seiji Nishino, Robin Dean and Lewanne Sharp for their assistance in sample preparation; and Michael Gelb for his assistance in manuscript preparation. REFERENCES 1 Bloom, F.E., The fine structural localization of biogenic monoamines in neural tissue, hu. Neurobiol., 13 (1970) 27-66. 2 Bradford, H.F., Jones, D.G. and Booher, J., Biochemical and morphological studies of the short and long term survival of isolated nerve-endings, Brain Res., 90 (1975) 245-259. 3 Baker, T.L. and Dement, W.C., Canine narcolepsy-cataplexy syndrome: evidence for an inherited monoaminergic-cholinergic imbalance. In D.J. Mc Ginty (Ed.), Brain Mechanisms of Sleep, Raven, New York, 1985, pp. 199-234. 4 Dodd, P.R., Hardy, J.A., Oakley, A.E. and Strong, A.J., Synaptosomes prepared from fresh human cerebral cortex; morphology, respiration and release of transmitter amino acids, Brain Res., 224 (1981) 419-425. 5 Drapeau, P., Long term storage of functional, isolated nerve endings by slow freezing and rapid thawing, J Neurosci. Methods, 24 (1988) 11-115. 6 Faull, K.F., DeAmicic, L.E., Radde, L., Bowersox, S.S., Baker, T.L., Kilduff, T.S. and Dement, W.C., Biogenic amine concentrations in the brains of normal and narcoleptic canines: current status, Sleep, 9 (1986) 107-110. 7 Garey, R.E. and Heath, R.G., Uptake of catechol~mines by human synaptosomes, Brabz Res., 79 (1974) 520-523. 8 Hardy, J.A. and Dodd, P.R., Metabolic and functional studies on post-mortem human brain, Neurochem. Int., 5 (1983) 253-266. 9 Hardy, J.A., Dodd, P.R., Oakley, A.E., Perry, R.H., Edwardsom, J.A. and Kidd, A.M., Metabolically active synaptosomes can be prepared from frozen rat and human brain, J. Neurochem., 40 (1983) 608-614. 10 Johnson, M.K., The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondrial preparations, Biochem. J., 7 (1960) 610-618. I1 Johnson, M.K. and Wittaker, V.P., Lactate dehydrogenase as a cytoplasmic marker in brain, Biochem. J., 88 (1963) 404-409. 12 Lim, R.K.S., Lin, C.N. and Moffit, R.F., A StereotaxicAtlas of the Dog's Brain, Thomas, Springfield, IL, 1960. 13 Mefford, I.W., Baker, T.L., Boehme, R., Foutz, A., Ciaranello, R., Barchas, J. and Dement, W.C., Narcolepsy: biogenic amine deficit in an animal model, Science, 220 (1983) 629-632. 14 Mignot, E., Guilleminault, C., Dement, W.C. and Grumet, F.C., Genetically determined animal models of narcolepsy, a disorder of REM sleep, in P. Driscoll (Ed.), Genetically-defined Animal Models of Neurobehavioral Dysfunction, Birkhaiiser Boston Inc., Cambridge, 1992, pp. 90-110. 15 Miller, J.D., Faull, K.F., Bowersox, S.S. and Dement, W.C., CNS monoamines and their metabolites in canine narcolepsy: replication study, Brain Res., 509 (1990) 169-171. 16 Olivera, L., Burns, A. and Bisalfutra, Y., The use of an ultra low medium (VCD/HXSA) in the rapid embedding of plant cells for electron microscopy, J. Microsc., 132 (1983) 195-202. 17 Schwarcz, R., Effects of tissue storage and freezing on brain glutamate uptake, Life Sci., 28 (1981) 1147-1154.
115
BRES 17982
Monoaminergic uptake in synaptosomes prepared from frozen brain tissue samples of normal and narcoleptic canines D e l p h i n e V a l t i e r , W i l l i a m C. D e m e n t a n d E m m a n u e l M i g n o t Stanford Unicersity, School of Medicine, Sleep Disorders Center, Palo Alto, CA 94304 (USA) (Accepted 17 March 1992)
Key words: Synaptosome; Frozen tissue; Noradrenaline; Dopamine; Serotonin uptake; Narcolepsy; REM sleep
Canine narcolepsy, a model of the human disorder, is associated with altered catecholamine but not serotonin (5-HT) metabolism in some brain areas, particularly the amygdala. A possible explanation for these global changes could be the existence of specific defects in monoamine uptake processes. We have studied the uptake of ['~Hlnorepinephrine (NE), [3H]dopamine (DA) and [3H]5-HT in synaptosomes prepared from cortex and amygdala of narcoleptic and control Doberman pinscher brains. Since narcoleptic canines are relatively few in number, we have used a specific brain freezing procedure that has been reported to allow restoration of metabolically functional tissue upon thawing. Preliminary studies comparing monoamine uptake in fresh and frozen brain samples of both groups of dogs were carried out and demonstrated that this procedure significantly altered serotoninergic but not noradrenergic and dopaminergic uptake. All further investigations were then done on synaptosomes prepared from frozen samples. Our results demonstrate that synaptosomal uptake of [3HINE, [3H]DA and [3H]5-HT in cortex and amygdala are not altered in narcolepsy.
INTRODUCTION
Narcolepsy is a genetically determined REM sleep disorder of unknown origin affecting humans and animals 3'4. At Stanford University, we have established a colony of Doberman pinschers that transmit the disease as a fully penetrant autosomal recessive trait. This model has been used extensively to determine the neurochemical basis of narcolepsy and the most consistently found abnormality reported (two independently replicated studies) has been an increased concentration of DOPAC and dopamine (DA) in the amygdala6'13'~5. In this study, we hypothesized that a possible explanation for this modification could be altered DA uptake in this brain region. In order to test this hypothesis, we have measured monoamine uptake in synaptosomes prepared from frozen control and narcoleptic brain structures. Synaptosomes are artificially prepared nerve endings that are able to accumulate neurotransmitters against a concentration gradient ~. Monoamine uptake that can be mea-
sured in synaptosomes is a specific, energy dependent process which is sensitive to metabolic inhibitors, temperature, ion concentration and various neuropharmacological compounds 7. It is therefore a suitable model that has been used by numerous investigators. Metabolically viable synaptosomes usually need to be prepared from fresh tissue since cellular respiration and structural organization of nerve endings need to be preserved. In this study, we have used a method that allows us to prepare active synaptosomes from tissue that has been slowly frozen and rapidly thawed 4'5's. This technique has been used successfully on human and animal tissue to study monoamine uptake ~7 and it allowed us to gather age-matched control and narcoleptic dog brains. Our results indicate that this technique can be successfully used to prepare active synaptosomes from frozen dog brains. We also showed that monoamine uptake of DA, norepinephrine (NE) and serotonin (5-HT) is not altered in cortex and amygdala of the narcoleptic dog.
Correspondence: E. Mignot, Stanford University, School of Medicine, Sleep Disorders Center, 701 Welch Road, Suite 2226, Paio Alto, CA 94304, USA.
116 MATERIALS A N D M E T H O D S
Subj¢cl$ Twelve Doberman pinschers of the Stanford Canine Narcolepsy Colony were included in the study. Animals were individually housed in the Division of Laboratory Animal Medicine of Stanford University. All experiments were performed in accord with the current edition of the NIH guide for the care and use of laboratory animals. Six animals were narcoleptic (born from two narcoleptic parents: 2 females and 4 males, aged 21.83 +_7.10 months) and had shown clear, spontaneous cataplectic and drug-induced attacks during the Food Elicited Cataplexy Test (FECT). The 6 other animals were control dogs (born from two normal parents: 2 females and 4 males, aged 23.66___4.25 months). Narcoleptic and control animals were born from 4 and 3 different crosses respectively in order to avoid litter effects in the comparison. Dogs were drug free for at least 2 weeks at the time of the sacrifice,
Tissue collection and storage procedure One control and one affected animal (roughly age-matched) were sacrificed the same day using an i.e. overdose of thiopental (60 mg/kg). Brains were quickly removed and the amygdala and frontoparietai cortex anatomically dissected at room temperaturel2. Brain pieces were weighed, individually placed in eppendorf tubes and frozen according to Hardy et al.'~. Tubes were collected in a plastic bag, wrapped in cotton wool, placed at -20°C for 8-9 h, then transferred to -70°C. A thermistance probe was inserted in one of the brain pieces to record local tissue temperature during the freezing procedure. When tissue had reached -70°C, samples were placed in a normal Revco box until use. On the day of the experiment, frozen tissue was rapidly thawed by immersion of the tube in a 37°C waterbath for 15-20 s (per 100 mg of tissue) and transferred immediately to an ice-cold 0.32 M sucrose, 5 mM Na-HEPES (N-2hydroxethyl piperazine-N-2-ethanesulfonic acid) pH 4 buffer for synaptosomal preparation.
Synaptosomal preparation Brain pieces were homogenized at 0°C using a poter teflon-glass homogenizer (12 strokes, 800 rpm, clearance 0.5 mm) and then crude homogenate centrifuged (Beckman L8 ultracentrifuge, fixed angel rotor Ti 70.1) at 5,000 rpm (w:t = 1.57.107) at 4°C. The resulting supernatant fraction was layered onto half of its volume of 1.2 M sucrose solution and centrifuged at 50,(100 rpm (w:t = 1.610~) to yield at the sucrose interface a crude fraction that contained synaptosomes, myelin and microsomes, This fraction was carefully collected, diluted in ice-cold 0.32 M sucrose, 5 mM HEPES solution, layered onto half of its volume of (1.8 M sucrose solution and centrifuged again under the same conditions (50,000 rpm, w2t = 1.610s) to generate a pellet enriched in synaptosomes. The pellet was then resuspended in 1 ml of 0.32 M sucrose, 5 mM HEPES/100 mg of original tissue to reach a concentration of 0.4-0.5 mg of proteins/ml and kept on ice for no longer than 3 h. Protein concentration was determined according to the Bradford technique:.
Synaptosomal [ 3Hlneurotransmitter uptake Optimum assay conditions for monoamine uptake were determined by studying the time-course of [3H]NE accumulation at different protein concentrations, 0.1-0.7 mg protein/ml. Accumulation of ['~H]NE into purified synaptosome preparation was linear during the initial 5 min of the incubation at 37°C. The initial uptake also shows a linear relationship with the protein concentrations (synaptosome concentrations) in the range of 0.1-0.5 mg protein/ml. Therefore, in the subsequent studies an incubation time of 5 rain at a final synaptosome concentration of 0.4-0.5 mg protein/ml was employed.
K M (the apparent affinity constant of the transporter) and Vmax (the maximum velocity of the transporter system) were determined for each experiment. Aliquots of the synaptosome-enriched homogenates were incubated in a buffer (pH 7.4) containing (in mM): NaCi 140, KCi 5, MgCI 2 I, glucose 10, HEPES 10 with ascorbic acid 0.5, disodium EGTA 0.1 and pargyline 0.05 Ca monoamine oxydase inhibitor). Fifty /.tl of homogenate was added at 0°C to 400 p,i of the incubation buffer. After equilibrating at 37°C for 10 min, 50 ~l of [3H]neurotransmitter was added to yield a final concentration ranging from 0.002 to 0.5 gM ([3H]NE, [3H]DA, [3H]5-HT). The synaptosomal suspensions were then incubated an additional 5 min to determine monoamine uptake. Non-specific uptakes were determined at 0°C. For [3H]DA and ['~H]5-HT, filters were pretreated with polyethyleneimine (0.1%) for 3-5 min before filtration. To stop the incubation, 5 ml of ice-cold uptake buffer was added in each tube and synaptosomes were separated by filtration on G F / C glass fiber filters (Whatman) with moderate vacuum, then filters were washed twice with 5 ml of ice-cold uptake buffer to eliminate unincorporated [3H]neurotransmitter. Filters were dissolved in scintillation mixture (Cytoscint) for liquid scintillation counting in a Beckman counter (Model LS 3801).
Electron microscopy in order to assess the quality of the synaptosomal preparations, all samples were examined using electron microscopy. Synaptosomes were incubated at 37°C for 30 min and pelleted in a bench centrifuge at room temperature (10,000 rpm for 2 rain). The pellet was then fixed by immersion in phosphate buffer (0.1 M pH 7.24) containing glutaraldehyde (2%) and paraformaldehyde (!%) overnight at 0°C and postfixed the following day in 1% osmium tetroxide. Samples were then embedded in a plastic mixture (VCD-HXSA) I~ consisting of vinylcyclohexylene silicone 200 (0.5 ml) and dimethyl-amino ethanol (0.5 ml). Sections were cut using a LKB ultramicrotome and viewed on a Philips 410 electron microsope. Pictures of diverse magnifications were taken to assess the purity of the synaptosomal fraction and the integrity of synaptosomal structures.
Lactate dehydrogenase assay The synaptosome concentration in the preparation was also evaluated by determining lactate dehydrogenase (LDH) activity. LDH is a cytoplasmic marker ~, exclusively present in synaptosomes but not mitochondria or other contaminating fragments. Total synaptosome lactate dehydrogenase activity was assayed using the method of. Johnson I°. LDH activity was expressed in international units per ml (amount of LDH that causes a decrease of 0.001 per minute in the absorbance at 340 nm of the 3 ml reaction mixture in a I cm curet).
Data analysis and statistics K M and Vmax of the uptake transporter were estimated using a linear regression after Lineweaver-Burk transformation, Values reported are means :t:S.E.M. of independent kinetic experiments. Statistical analysis was performed using SYSTAT, Inc. (Evanston, IL) on a Macintosh SE.
Drugs The following substances were used: [ethyl-l-3H(N)dopamine (spec. act. 37 Ci/mmol; L[7-3H]noradrenaline (spec. act. 14.3 Ci/mmol) and 5-hydroxy['~H]tryptamine creatinine sulphate (spec. act. 21 Ci/mmol) NEN Boston, MA. Ascorbic acid, pargyline, disodium EDTA (ethylenglycoi-bis-aminoethylether-N,N'-tetracetic acid) and LDH diagnostic kit NADH were purchased from Sigma (St Louis, MO).
Fig. 1. Electron microscopy photographs of synaptosomes prepared from slowly frozen/fast thawed amygdala samples of control (A) and narcoleptic (B) dog brains. Bar = 4 ~m.
lC
!ill
118 TABLE !
TABLE !I
Comparison of ['~H]NE, [~H]DA and ['~H]5-HT uptake in s),naptosomes prepared from fresh and slowly frozen/fiast thawed cortical samples
Comparison of I SH]NE, [ 3H]DA and [ 3H]5-HT uptake in synaptosomes prepared from slowly frozen ~fast thawed cortical samples from narcoleptic (N) and control (C) dogs
K M values are given in 10 -7 M, I/max values in pmol/mg protein/rain. Values are means + S.E.M. of 6 samples for [3H]NE uptake and of 3-4 samples for [3H]DA and [3H]5-HT uptake. No slatisticai differences were observed using a Student's t-test except for ['aH]5-HT uptake (Vmax, P < 0.001).
K M values are given in 10 -7 M, Vmax values in p m o l / m g protein/ min. Values are means +-S.E.M. of 6 samples for [3H]NE uptake and samples for [3H]DA and [3H]5-HT uptake. No statistical differences were observed using a Student's t-test or a non-parametric Mann-Whitney U-test.
['~H]NE
[ 3H]DA
13H]NE
['~H]5-HT
I';H]DA
[ 3H]5-HT
KM
V,,, ,
KM
V,.....
K M
V,....
KM
V,.a.~
KM
V,,,~x
KM
V,,,.,
Fresh
2.82 + 1.07
0.775 +0.158
2.76 + 2.16
I. ! 63 +0.575
0.539 +_0.05
1.917 +_0.095
C
0.580 + 0.156
0.713 + 0.353
0.756 + 0.282
4.706 + 3.00
1.96 + 0.982
1.144 + 0.573
Frozen
2.39 +_0.74
0.656 +0.162
1.69 0.731 1.08 +_0.62 +_0.202 +_0.27
0.776 +_0.065
N
0.961 +0.127
0.577 +0.226
0.912 +0.530
3.40 + 1.67
1.13 +-0.83
0.956 +-0.884
RESULTS
Effects of freezing and thawing on the synaptosomal preparation Using electron microscopy, no morphological differences were found between synaptosomes prepared from fresh or frozen control and narcoleptic dog cortex brain samples (data not shown). Synaptosomal fractions (membrane-bound vesicle containing structures) were reasonably homogenous and uncontaminated by mitochondria (laminar apperance) or myelin and broken membranes (less than 50% contamination). Synaptosome sizes (diameters) ranged between 0.6 and 0.9 ~m. Electron microscopic analysis of the slowly frozen/fast thawed amygdala preparations did not show differences between narcoleptic and control dog brains. LDH activity was also determined in all the synaptosomal samples tested. No significant differences were found between values obtained in fresh or frozen samples (control or narcoleptic, cortex or amygdala).
Effects of freezing and thawing on synaptosomal uptake of monoamines The effect of the freezing/thawing procedure was explored using the 3 monoamines. Results are presented in Table I. A tendency to a decreased Vmax was observed in frozen samples for all monoamines but was statistically significant only for serotonin uptake. No significant changes in KM were observed when fresh or slowly frozen/fast thawed canine cortex tissue was t,sed. All subsequent experiments were carried out using slowly frozen/fast thawed tissues.
Morphological differences in synaptosomes obtained from control and narcoleptic animals Protein and LDH concentrations in amygdala preparations did not differ between narcoleptic and
control samples. For controls and narcoleptics values were respectively: 0.438 + 0.064 mg protein/ml, 0.481 + 0.050 mg protein/ml (Student's t-test, P > 0.297) and 1077.5 + 208.6 LDH units; 1032.5 + 175.4 LDH units (Student's t-test, P > 0.464). In both groups, a significant correlation between protein concentrations and LDH units was observed, thus indicating that the same proportion of synaptosomes were present in both preparations (Controls, r = 0.790 and Narcoleptics, r = 0.903). Electron microscopy performed on the samples also confirmed the first results observed in the cortical preparations. No major alterations were found in either narcoleptic or control dog tissues after slow freezing and rapid thawing. Fig. 1A, B represents electron microscopy photographs of synaptosomes prepared from the amygdala of control and narcoleptic dogs respectively.
Investigation of uptake parameters in narcolepsy Uptake of [aH]NE, [aH]DA and [aH]5-HT was assessed in synaptosomes prepared from cortex and amygdala. Uptake parameters for the 3 radioligands TABLE III
Comparison of 13H]NE, [';H]DA and 13H]5.HT uptake in synaptosprees prepared from slowly frozen/fast thawed amygdala samples from narcoleptic and control dogs. K M values are given in 10 -7 M, Vmax values in p m o l / m g protein/ rain. Values are means +_S.E.M. of 6 samples for [3H]NE and [aH]DA uptake and 3 samples for [3H]5-HT uptake. No statistical differences were observed using a Student's t-test or a non-parametric MannWhitney U-test.
[SHINE
['*H]DA
l SHI5-HT
KM
V,,ax
KM
V,mx
C
2.15 +_0.66
0.397 +_0.105
1.81 +_0.34
0.509 1.76 +_0.082 +_0.26
KM
1.55 +_0.47
N
1.50 +_0.37
0.409 +_0.124
1.00 5:0.307
0.482 +-0.128
1.32 +_0.25
2.13 _+0.85
E, ax
119
were investigated on the same tissue preparation, each experiment consisting of a simultaneous analysis of a control and a narcoleptic dog sample. No statistical differences were found between narcoleptic and control dogs for the kinetic uptake parameters obtained for the three neurotransmitters in cortex or amygdala (Tables II and Ill). DISCUSSION
One of the most consistent neurochemical abnormalities reported in the brain of narcoleptic canines is a change in DA content and metabolism in the amygdala 6a3'~5. In this study, we have investigated the uptake of monoamines in synaptosomes prepared from the amygdala of narcoleptic and control dogs. No difference was observed, suggesting that the neurochemical changes reported in previous studies are not secondary to abnormal uptake processes in this structure. In addition, electron microscopy on the samples did not reveal any gross morphological abnormalities in the synaptosomal vesicular formation. An interesting aspect of this study is the use of slowly frozen/rapidly thawed tissue 9, instead of fresh tissue to assess synaptosomal function. This technique is particularly useful if tissue samples are scarce since it affords the possibility of performing several independent studies on the same brain. This technique has been used in rodents where the brain could be processed immediately after death and in humans where variable lag between death and processing always has to be taken into account in the interpretation of the result, in this study, we demonstrated that this technique is also practical for another large-brain-sized mammal, the dog. Using this species, our protocol demonstrated only a very limited difference in monoamine uptake and morphology between fresh and frozen preparations. In conclusion, this study demonstrates the usefulness of the slow freezing/rapid thawing technique to prepare viable synaptosomal preparations in species where resources are scarce. Using this technique, we could not observe any changes in monoamine uptake parameters in the amygdala of the narcoleptic dog.
Acknowledgements. This work was supported by Grant NIH NS 23724 to W.C.D. We thank Fran Thomas for her participation in the electron microscopy studies; Seiji Nishino, Robin Dean and Lewanne Sharp for their assistance in sample preparation; and Michael Gelb for his assistance in manuscript preparation. REFERENCES 1 Bloom, F.E., The fine structural localization of biogenic monoamines in neural tissue, hu. Neurobiol., 13 (1970) 27-66. 2 Bradford, H.F., Jones, D.G. and Booher, J., Biochemical and morphological studies of the short and long term survival of isolated nerve-endings, Brain Res., 90 (1975) 245-259. 3 Baker, T.L. and Dement, W.C., Canine narcolepsy-cataplexy syndrome: evidence for an inherited monoaminergic-cholinergic imbalance. In D.J. Mc Ginty (Ed.), Brain Mechanisms of Sleep, Raven, New York, 1985, pp. 199-234. 4 Dodd, P.R., Hardy, J.A., Oakley, A.E. and Strong, A.J., Synaptosomes prepared from fresh human cerebral cortex; morphology, respiration and release of transmitter amino acids, Brain Res., 224 (1981) 419-425. 5 Drapeau, P., Long term storage of functional, isolated nerve endings by slow freezing and rapid thawing, J Neurosci. Methods, 24 (1988) 11-115. 6 Faull, K.F., DeAmicic, L.E., Radde, L., Bowersox, S.S., Baker, T.L., Kilduff, T.S. and Dement, W.C., Biogenic amine concentrations in the brains of normal and narcoleptic canines: current status, Sleep, 9 (1986) 107-110. 7 Garey, R.E. and Heath, R.G., Uptake of catechol~mines by human synaptosomes, Brabz Res., 79 (1974) 520-523. 8 Hardy, J.A. and Dodd, P.R., Metabolic and functional studies on post-mortem human brain, Neurochem. Int., 5 (1983) 253-266. 9 Hardy, J.A., Dodd, P.R., Oakley, A.E., Perry, R.H., Edwardsom, J.A. and Kidd, A.M., Metabolically active synaptosomes can be prepared from frozen rat and human brain, J. Neurochem., 40 (1983) 608-614. 10 Johnson, M.K., The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondrial preparations, Biochem. J., 7 (1960) 610-618. I1 Johnson, M.K. and Wittaker, V.P., Lactate dehydrogenase as a cytoplasmic marker in brain, Biochem. J., 88 (1963) 404-409. 12 Lim, R.K.S., Lin, C.N. and Moffit, R.F., A StereotaxicAtlas of the Dog's Brain, Thomas, Springfield, IL, 1960. 13 Mefford, I.W., Baker, T.L., Boehme, R., Foutz, A., Ciaranello, R., Barchas, J. and Dement, W.C., Narcolepsy: biogenic amine deficit in an animal model, Science, 220 (1983) 629-632. 14 Mignot, E., Guilleminault, C., Dement, W.C. and Grumet, F.C., Genetically determined animal models of narcolepsy, a disorder of REM sleep, in P. Driscoll (Ed.), Genetically-defined Animal Models of Neurobehavioral Dysfunction, Birkhaiiser Boston Inc., Cambridge, 1992, pp. 90-110. 15 Miller, J.D., Faull, K.F., Bowersox, S.S. and Dement, W.C., CNS monoamines and their metabolites in canine narcolepsy: replication study, Brain Res., 509 (1990) 169-171. 16 Olivera, L., Burns, A. and Bisalfutra, Y., The use of an ultra low medium (VCD/HXSA) in the rapid embedding of plant cells for electron microscopy, J. Microsc., 132 (1983) 195-202. 17 Schwarcz, R., Effects of tissue storage and freezing on brain glutamate uptake, Life Sci., 28 (1981) 1147-1154.