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REM sleep deprivation increases the levels of tyrosine hydroxylase and norepinephrine transporter mRNA in the locus coeruleus
Molecular Brain Research 57 Ž1998. 235–240
Research report
REM sleep deprivation increases the levels of tyrosine hydroxylase and norepinephrine transporter mRNA in the locus coeruleus Radhika Basheer, Meredith Magner, Robert W. McCarley, Priyattam J. Shiromani
)
VA Medical Center and HarÕard Medical School, 940 Belmont St., Brockton, MA 02401, USA Accepted 24 March 1998
Abstract The present study was conducted to determine the effects of REM sleep deprivation on the levels of tyrosine hydroxylase ŽTH. and norepinephrine transporter ŽNET. mRNA in the locus coeruleus ŽLC. of rats. The animals were deprived of REM sleep for 1, 3 or 5 days, then killed and changes in the mRNA levels were determined using in situ hybridization. The levels of both TH and NET mRNA increased in animals deprived of REM sleep for 3 days or longer whereas no change in these messages were observed in the LC of control animals. REM sleep deprivation has been used as a mode of treatment for major depression. Others have shown that treatment with tricyclic antidepressants also results in increased levels of TH and NET mRNA in LC. Our results suggest that the antidepressant effect of REM sleep deprivation and tricyclic antidepressants may share similar molecular changes in the norepinephrine system. q 1998 Elsevier Science B.V. All rights reserved. Keywords: REM sleep deprivation; Tyrosine hydroxylase; Norepinephrine transporter; In situ hybridization; Locus coeruleus
1. Introduction In rats, the locus coeruleus ŽLC. consists principally of norepinephrine ŽNE. containing neurons which project to many brain regions w3,27x. The discharge activity of these neurons is highest during waking, decreases during slow wave sleep and is virtually silent during REM w2,6,9x. In the absence of REM sleep these neurons fire continuously resulting in the reduction in receptor sensitivity w18,19,35x and changes associated with NE synthesis and metabolism w28,29x. In rats, selective REM sleep deprivation causes a significant increase in NE levels in the lateral hypothalamus w4x. Turnover and metabolism of NE remains elevated during and after REM sleep deprivation w28,29,33x. The activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is high in the whole brain after 96 h of REM deprivation w38x. Total sleep deprivation for 52 h also increases TH activity in the hypothalamus w26x. Increased TH mRNA is noted in the LC in rats in response to 3 days of REM sleep deprivation w28x.
) Corresponding author. VA Medical Center and Harvard Medical School, Research 151C, 940 Belmont Street, Brockton, MA 02401, USA. Fax: q1-508-895-0002; E-mail: [email protected]
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 8 8 - 6
The norepinephrine transporter ŽNET. w17x is another aspect of the NE system that is likely to be affected as a result of REM sleep deprivation-induced activation of the LC. Neurotransmitter transporters are subject to regulatory mechanisms that affect synaptic efficacy w12,16x. As a membrane protein, NET is responsible for reuptake and maintenance of NE stores at the synapse w8x. The levels of NET have been shown to be up or downregulated in response to increased or decreased NE release w15x. Although other aspects of the NE system have been studied following REM sleep deprivation, the effects on NET are unknown. Therefore, this study examined the effects of REM sleep deprivation on the levels of TH and NET mRNA in the LC.
2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats obtained from Charles River Lab were placed in a room with 12:12 h light dark cycle Žlights on 0700 h:off 1900 h. and controlled temperature Ž258C..
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2.2. Sleep depriÕation paradigm Rats Žaverage weight 345 " 2.86 g. were deprived of REM sleep by placing them on a small platform Ž9 cm in diameter. in the middle of a tub filled with water Ž3 in. deep.. This method is effective in inducing REM sleep deprivation because when the rats enter into REM sleep there is a loss of tone in antigravity muscles which makes them fall into the water and awaken. Control rats were placed on larger diameter platforms Ž12 cm in diameter. which exposed them to the same experimental environment as rats placed on small platforms but without the REM sleep deprivation. To lessen the stress associated with social isolation, two rats were placed in a tub containing three platforms. The temperature of the water in the tub was 308C and was changed daily. Food and water were available ad libitum throughout the time the rats were on the platforms. The animals were weighed before and after placement on the platform. There was no significant change in the weight as a result of small platform ŽPre SP 345 " 3.59; Post SP 343.3 " 5.95. or large platform ŽPre LP 345 " 5.0; Post LP 350 " 6.5. treatment. Previously, we have demonstrated that the size of the small platform Ž9 cm diameter. is effective in depriving animals of REM sleep w31x. The rats were on platforms for 1 or 3 or 5 days. Seven experimental groups were used. Ž1. Large platform for one day ŽLP1.; Ž2. five days ŽLP5.; Ž3. small platform for one day ŽSP1.; Ž4. three days ŽSP3.; Ž5. and five days ŽSP5.; Ž6. three days on SP and then transferred for 2 days on LP ŽSP3 q LP2.; Ž7. dry cage controls. At the end of the relevant time period on platforms the animals were killed by using an overdose of Nembutal Ž200 mgrkg; IP., perfused transcardially with 50 ml of ice cold normal saline followed by 350 ml of ice cold 2% formaldehyde in 0.1 M phosphate buffer saline ŽPBS.. The brains were removed, placed overnight in the same fixative, transferred and stored in buffer containing 20% sucrose, 0.1 M PBS, pH 7.4 at 48C for immunohistochemistry or in situ hybridization.
2.3. In situ hybridization The TH probe was a 30 bp long oligonucleotide Žobtained from New England Nuclear, NEN.. It was end labeled using 35 S-dATP with a 3X end labeling kit ŽNEP 100.. The reaction conditions and purification of the probe was performed as per the manufacturer’s instructions. The NET clone was a kind gift from Richard Simerly. The 35 S-UTP labeled riboprobe complementary to NET mRNA was synthesized using T3 polymerase ŽPromega. to transcribe a 660 bp insert in Bluescript SK-II plasmid. Coronal sections Ž14 m m thick. were cut on a cryostat and thaw mounted on gelatin coated slides. Sections were
fixed in 4% paraformaldehyde and PBS ŽpH 7.4. for 10 min, washed twice with cold PBS, acetylated in 0.15% triethanolamine containing 2% acetic anhydride for 10 min, washed twice in PBS and dehydrated using 70%, 90% and 100% ethanol and dried briefly. Sections were prehybridized for 1 h at 378C in buffer containing 50% formamide, 5 = PIPES, 1 = Denhardts, 0.2% SDS, 40 mM DTT, 250 mgrml salmon sperm DNA Žsheared.. For hybridization purified radioactive probe Ž3 = 10 6 cpmrslide. was added to the above mentioned prehybridization solution along with 1% yeast tRNA ŽNEN.. Each slide contained 3–4 sections. 200 m l of hybridization solution was added and the slides sealed with leakproof coverslips ŽProbeclip PC 200, Grace. and incubated at 378C overnight. The slides were washed with 4 = SSC Ž1 = SSC s 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. containing 10 mM of b-mercaptoethanol followed by a wash using RNase Ž10 mgrml. for 15 min at room temperature. This was followed by 20 min washes with increasing salt stringency ending with 0.5 = SSC at 558C. Slides were dried and exposed to film ŽKodak X-OMAT. for a week. A Leitz Aristoplan microscope was used to view the intensity of labeling in the sections and the radioactive signal in LC was measured using the image analysis system ŽNIH Image version 1.56.. 2.4. c-Fos immunohistochemistry c-Fos-ir cells were detected using the protocol described previously w36x. Briefly a rabbit anti-Fos antibody ŽSanta Cruz. was used at a dilution of 1:1000. The sections Ž40 m m thick. were washed in 0.1 M PBS containing 0.3% H 2 O 2 followed by another wash with 0.1 M PBS containing 0.25% Triton-X solution, and then incubated overnight at room-temperature in the antibody. The following day the sections were washed twice in 0.1 M PBS and incubated for 1 h with a biotinylated anti-rabbit secondary antibody. After washing, the sections were incubated for 1 h in the avidin–biotin–peroxidase ŽVector. complex. Subsequently, diaminobenzidene ŽDAB. with nickel chloride intensification was used as a chromogen to visualize the reaction products. The sections were then mounted onto gelatin-coated slides and coverslipped after dehydration in alcohol–xylene solutions. A Leitz Aristoplan microscope was used to obtain photomicrographs of LC and Barrington’s nucleus. 2.5. Counts of labeled nuclei In four rats per group, the regional expression of Fos-ir in Barrington’s nucleus Žleft side. was determined by counting the labeled nuclei Žone in four series of sections. in three consecutive sections. The individual counting the cells was blind to the groups.
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2.6. Statistical analysis One-way ANOVA was used for data analysis. Post-hoc analysis with Bonferroni adjustment for multiple comparisons was used to compare the experimental groups Žanimals on small and large platforms. against the dry cage control group.
3. Results 3.1. TH mRNA leÕels Rats were deprived of REM sleep for 1, 3 or 5 days by utilizing the platform method of REM sleep deprivation Žsee Section 2.. TH mRNA levels were determined after 1, 3 or 5 days on platforms. A one-way ANOVA revealed significant difference between groups in TH mRNA levels Ž F s 5.181; df s 6.46; p - 0.001.. Post-hoc analysis using Bonferroni adjustment for multiple comparisons revealed a significant increase in the levels of TH mRNA Žq32.6%; t s 4.99; df s 9; p - 0.001. in the LC of animals placed on small platform for 3 days when compared to dry cage controls ŽFig. 1A and B, Fig. 2A.. The mRNA levels remained high Žq55.4%; p - 0.003. after 5 days on small platform ŽFig. 2A.. On the other hand, the animals placed on large platform did not show any changes in TH mRNA levels compared to dry cage controls. Another set of animals were placed on small platform for 3 days and
Fig. 2. A significant increase in TH ŽA. and NET ŽB. mRNA was observed in rats placed on small platform for 3 ŽSP3. or 5 days ŽSP5.. Rats that were placed on small platform for 1 day ŽSP1. or large platform for 1 ŽLP1. or 5 days ŽLP5. did not show a significant increase in these message levels. Rats that were placed on small platforms for 3 days and then placed on large platforms for 2 days ŽSP3qLP2. also did not show any changes in TH or NET mRNA. Asterisk Ž). denotes significance compared to dry cage ŽDC. control rats.
Fig. 1. Autoradiograph depicting changes in the levels of TH and NET mRNA in the locus coeruleus. Coronal sections show that TH labeling in LC of rats deprived of REM sleep for 3 days ŽSP3. is increased ŽB. when compared to rats that were not REM sleep deprived Žthe dry cage controls, DC. ŽA.. Similarly the NET mRNA levels also increased with 3 days of REM sleep deprivation ŽSP3. animals ŽD. when compared to dry cage controls ŽC..
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transferred to large platform for 2 days. This was done in order to determine whether the changes in the TH mRNA levels in these animals were due to the REM sleep deprivation induced by the small platform and not due to the platform placement itself. In these animals the TH mRNA levels returned to baseline levels Ždry cage. indicating that when animals are placed on large platform they are able to obtain REM sleep ŽFig. 2A.. 3.2. NET mRNA leÕels The changes in the NET mRNA levels were significant as assessed by overall analysis of variance Ž F s 4.29; df s 6.14; p - 0.012.. Post-hoc analysis showed that animals placed on small platform for 3 days had a significant increase in NET mRNA Žq20.8%; t s 5.25; df s 4; p 0.006. when compared to dry cage controls ŽFig. 1C and D, Fig. 2B.. Rats placed on small platform for 5 days continued to show an increase Žq17.9%; t s 7.892; df s 4; p - 0.001. in NET mRNA levels ŽFig. 2B.. The levels of NET mRNA returned to baseline values when the animals
Fig. 4. The number of c-Fos-positive neurons in the Barrington’s nucleus were significantly higher Ž p- 0.05. after one day on both large and small platform animals when compared to the dry cage controls. After 5 days on platforms Fos immunoreactivity declined to control values. Four rats for each group were used.
were transferred to large platform for 2 days after being on small platform for 3 days ŽFig. 2B.. 3.3. Fos-ir in LC and Barrington’s nucleus The animals placed on either large or small platforms for 1 day showed increased numbers of c-Fos-ir neurons in Barrington’s nucleus. However with prolonged placement on the platforms few, if any, c-Fos-ir cells were found ŽFigs. 3 and 4.. Interestingly, in LC, c-Fos-ir neurons were not detected as a result of the REM sleep deprivation.
4. Discussion
Fig. 3. Photomicrographs of c-Fos-immunoreactive Žc-Fos-ir. neurons in the Barrington’s nucleus Žarrow.. c-Fos-ir neurons were evident in the nucleus of animals placed on small platform for one day Žphoto B. whereas both dry cage controls ŽA. and SP5 animals ŽC. did not show any c-Fos immunoreactivity.
This study examined TH and NET mRNA levels in the LC of rats deprived of REM sleep. The platform method was used to deprive the animals of REM sleep. The efficiency of the platform paradigm in inducing REM sleep deprivation was tested in our laboratory by determining the EEG and rebound sleep pattern after 1, 3 or 5 days on platforms w31x. Rats placed on small platforms but not on large platforms for 3 or 5 days showed an increased REM sleep rebound. This indicates that animals placed on small platforms but not on large platforms are deprived of REM sleep. The weights of the animals did not show any significant changes after 1, 3 or 5 days on platforms Ždata not shown.. Mendelson et al. w23x have shown that animals placed on small platforms for 4 days had a significant increase in REM sleep during the first 24 h of the rebound period. Similar increases in REM sleep have been reported after 2 days w24x or one day w30x on small platform. The TH and NET mRNA levels increased significantly after 72 h on small platform when compared to large platform and dry cage animals. These levels declined to
R. Basheer et al.r Molecular Brain Research 57 (1998) 235–240
control values when the animals were transferred to large platform for two days. Our results that TH mRNA levels are increased after REM sleep deprivation are in agreement with a previous report w28x describing similar effects after 72 h on small platform. In that study the animals were transferred to home cage for 24 h which resulted in a decrease of TH mRNA to control levels. In the present study, the increased TH and also NET mRNAs returned to baseline levels when the animals were transferred to LP after 72 h on small platform. These results indicate that increases in TH and NET mRNA levels are due to REM deprivation occurred due to the placement of rats on small platform. The changes in TH and NET mRNA might be due to the stress associated with the platform method of REM sleep deprivation. Increases in TH protein and mRNA have been reported to occur in response to several stress paradigms such as chronic heat exposure w7x, social stress w42x, isolation stress w1x or immobilization stress w20x. Stress mediated induction of corticotropin-releasing factor ŽCRF. increases neuronal activity in LC neurons. Such an activation is shown to result in increased c-fos mRNA expression w10x. Another group of neurons lying ventromedial to the LC known as Barrington’s nucleus, the micturition center, is also known to respond to CRF induction w40x. Both acute and chronic stress induce c-fos in Barrington’s nucleus, supporting the notion that this nucleus responds to stress w10,11x. It can be argued that animals placed on platforms Žeither on large or small. for one day experience acute stress while animals on the platforms for 3–5 days experience chronic stress. Previously it was shown that the serum corticosterone levels are high in animals placed on small and large platform for one day w28x. Our results show that c-Fos-ir in the Barrington’s nucleus was higher after one day on both small and large platform when compared to the dry cage controls and animals placed on platforms Žboth large and small. for 5 days. Collectively, these effects indicate that the animals experience stress during the first 24 h on the platform. However, the levels of TH and NET mRNA do not show any increase after one day on platforms. On the other hand, prolonged placement of animals on platforms is less likely to be stressful as they adapt to the new environment. Corticosterone levels have been shown to return to baseline levels after 72 h on large and small platform w28x. In the present study the number of c-Fos-ir neurons decreased in Barrington’s nucleus of animals placed on platforms Žeither small or large. for 5 days. The REM sleep rebound is increased only in animals placed on small platform for 5 days w31x. This suggests that the increases in TH and NET mRNA is not due to stress but is due to REM sleep deprivation. Interestingly, c-Fos-ir was not seen in LC at any tested time points. These findings may be clinically relevant to major depressive disorder. REM sleep deprivation has been shown
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to alleviate major depression w21,41,43x and we have suggested that the treatment acts by increasing the availability of monoamines at brain synapses w22x. Brain noradrenergic mechanisms are hypothesized to contribute to major depressive disorder both in humans w45x and in rat models of depression w25,44x. Reduction in norepinephrine ŽNE. levels have been suggested to result in major depression w5,13,32,34x. Tricyclic antidepressants and monoamine oxidase inhibitors increase availability of NE at the synapse and are used to treat depression. It is conceivable that the decrease in NE activity might also be associated with changes in norepinephrine transporter. In fact, recently it was shown that NET in the locus coeruleus ŽLC. is reduced in humans with major depression w14x. Our results show that REM sleep deprivation results in increased NET and TH mRNA levels. The antidepressant, desipramine, has been shown to increase NET mRNA in the LC w37,39x. This indicates that REM sleep deprivation and antidepressant treatments cause similar molecular changes in the NE system. The antidepressant effect of REM sleep deprivation might be due to increased activity of the LC neurons resulting in enhanced synthesis and release of NE at the synapse which in turn results in increased expression of the transporter. Collectively, these reports indicate that an upregulation of NET expression may contribute to alleviating depression.
Acknowledgements We would like to thank Katie Ryan and Ann-Marie Siegel for counting Fos-ir cells. This research was supported by VA Medical Research Service and NIH-NS 30140. Part of this work was presented at the 1996 annual meeting for Society for Neuroscience.
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Research report
REM sleep deprivation increases the levels of tyrosine hydroxylase and norepinephrine transporter mRNA in the locus coeruleus Radhika Basheer, Meredith Magner, Robert W. McCarley, Priyattam J. Shiromani
)
VA Medical Center and HarÕard Medical School, 940 Belmont St., Brockton, MA 02401, USA Accepted 24 March 1998
Abstract The present study was conducted to determine the effects of REM sleep deprivation on the levels of tyrosine hydroxylase ŽTH. and norepinephrine transporter ŽNET. mRNA in the locus coeruleus ŽLC. of rats. The animals were deprived of REM sleep for 1, 3 or 5 days, then killed and changes in the mRNA levels were determined using in situ hybridization. The levels of both TH and NET mRNA increased in animals deprived of REM sleep for 3 days or longer whereas no change in these messages were observed in the LC of control animals. REM sleep deprivation has been used as a mode of treatment for major depression. Others have shown that treatment with tricyclic antidepressants also results in increased levels of TH and NET mRNA in LC. Our results suggest that the antidepressant effect of REM sleep deprivation and tricyclic antidepressants may share similar molecular changes in the norepinephrine system. q 1998 Elsevier Science B.V. All rights reserved. Keywords: REM sleep deprivation; Tyrosine hydroxylase; Norepinephrine transporter; In situ hybridization; Locus coeruleus
1. Introduction In rats, the locus coeruleus ŽLC. consists principally of norepinephrine ŽNE. containing neurons which project to many brain regions w3,27x. The discharge activity of these neurons is highest during waking, decreases during slow wave sleep and is virtually silent during REM w2,6,9x. In the absence of REM sleep these neurons fire continuously resulting in the reduction in receptor sensitivity w18,19,35x and changes associated with NE synthesis and metabolism w28,29x. In rats, selective REM sleep deprivation causes a significant increase in NE levels in the lateral hypothalamus w4x. Turnover and metabolism of NE remains elevated during and after REM sleep deprivation w28,29,33x. The activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is high in the whole brain after 96 h of REM deprivation w38x. Total sleep deprivation for 52 h also increases TH activity in the hypothalamus w26x. Increased TH mRNA is noted in the LC in rats in response to 3 days of REM sleep deprivation w28x.
) Corresponding author. VA Medical Center and Harvard Medical School, Research 151C, 940 Belmont Street, Brockton, MA 02401, USA. Fax: q1-508-895-0002; E-mail: [email protected]
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 0 8 8 - 6
The norepinephrine transporter ŽNET. w17x is another aspect of the NE system that is likely to be affected as a result of REM sleep deprivation-induced activation of the LC. Neurotransmitter transporters are subject to regulatory mechanisms that affect synaptic efficacy w12,16x. As a membrane protein, NET is responsible for reuptake and maintenance of NE stores at the synapse w8x. The levels of NET have been shown to be up or downregulated in response to increased or decreased NE release w15x. Although other aspects of the NE system have been studied following REM sleep deprivation, the effects on NET are unknown. Therefore, this study examined the effects of REM sleep deprivation on the levels of TH and NET mRNA in the LC.
2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats obtained from Charles River Lab were placed in a room with 12:12 h light dark cycle Žlights on 0700 h:off 1900 h. and controlled temperature Ž258C..
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2.2. Sleep depriÕation paradigm Rats Žaverage weight 345 " 2.86 g. were deprived of REM sleep by placing them on a small platform Ž9 cm in diameter. in the middle of a tub filled with water Ž3 in. deep.. This method is effective in inducing REM sleep deprivation because when the rats enter into REM sleep there is a loss of tone in antigravity muscles which makes them fall into the water and awaken. Control rats were placed on larger diameter platforms Ž12 cm in diameter. which exposed them to the same experimental environment as rats placed on small platforms but without the REM sleep deprivation. To lessen the stress associated with social isolation, two rats were placed in a tub containing three platforms. The temperature of the water in the tub was 308C and was changed daily. Food and water were available ad libitum throughout the time the rats were on the platforms. The animals were weighed before and after placement on the platform. There was no significant change in the weight as a result of small platform ŽPre SP 345 " 3.59; Post SP 343.3 " 5.95. or large platform ŽPre LP 345 " 5.0; Post LP 350 " 6.5. treatment. Previously, we have demonstrated that the size of the small platform Ž9 cm diameter. is effective in depriving animals of REM sleep w31x. The rats were on platforms for 1 or 3 or 5 days. Seven experimental groups were used. Ž1. Large platform for one day ŽLP1.; Ž2. five days ŽLP5.; Ž3. small platform for one day ŽSP1.; Ž4. three days ŽSP3.; Ž5. and five days ŽSP5.; Ž6. three days on SP and then transferred for 2 days on LP ŽSP3 q LP2.; Ž7. dry cage controls. At the end of the relevant time period on platforms the animals were killed by using an overdose of Nembutal Ž200 mgrkg; IP., perfused transcardially with 50 ml of ice cold normal saline followed by 350 ml of ice cold 2% formaldehyde in 0.1 M phosphate buffer saline ŽPBS.. The brains were removed, placed overnight in the same fixative, transferred and stored in buffer containing 20% sucrose, 0.1 M PBS, pH 7.4 at 48C for immunohistochemistry or in situ hybridization.
2.3. In situ hybridization The TH probe was a 30 bp long oligonucleotide Žobtained from New England Nuclear, NEN.. It was end labeled using 35 S-dATP with a 3X end labeling kit ŽNEP 100.. The reaction conditions and purification of the probe was performed as per the manufacturer’s instructions. The NET clone was a kind gift from Richard Simerly. The 35 S-UTP labeled riboprobe complementary to NET mRNA was synthesized using T3 polymerase ŽPromega. to transcribe a 660 bp insert in Bluescript SK-II plasmid. Coronal sections Ž14 m m thick. were cut on a cryostat and thaw mounted on gelatin coated slides. Sections were
fixed in 4% paraformaldehyde and PBS ŽpH 7.4. for 10 min, washed twice with cold PBS, acetylated in 0.15% triethanolamine containing 2% acetic anhydride for 10 min, washed twice in PBS and dehydrated using 70%, 90% and 100% ethanol and dried briefly. Sections were prehybridized for 1 h at 378C in buffer containing 50% formamide, 5 = PIPES, 1 = Denhardts, 0.2% SDS, 40 mM DTT, 250 mgrml salmon sperm DNA Žsheared.. For hybridization purified radioactive probe Ž3 = 10 6 cpmrslide. was added to the above mentioned prehybridization solution along with 1% yeast tRNA ŽNEN.. Each slide contained 3–4 sections. 200 m l of hybridization solution was added and the slides sealed with leakproof coverslips ŽProbeclip PC 200, Grace. and incubated at 378C overnight. The slides were washed with 4 = SSC Ž1 = SSC s 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0. containing 10 mM of b-mercaptoethanol followed by a wash using RNase Ž10 mgrml. for 15 min at room temperature. This was followed by 20 min washes with increasing salt stringency ending with 0.5 = SSC at 558C. Slides were dried and exposed to film ŽKodak X-OMAT. for a week. A Leitz Aristoplan microscope was used to view the intensity of labeling in the sections and the radioactive signal in LC was measured using the image analysis system ŽNIH Image version 1.56.. 2.4. c-Fos immunohistochemistry c-Fos-ir cells were detected using the protocol described previously w36x. Briefly a rabbit anti-Fos antibody ŽSanta Cruz. was used at a dilution of 1:1000. The sections Ž40 m m thick. were washed in 0.1 M PBS containing 0.3% H 2 O 2 followed by another wash with 0.1 M PBS containing 0.25% Triton-X solution, and then incubated overnight at room-temperature in the antibody. The following day the sections were washed twice in 0.1 M PBS and incubated for 1 h with a biotinylated anti-rabbit secondary antibody. After washing, the sections were incubated for 1 h in the avidin–biotin–peroxidase ŽVector. complex. Subsequently, diaminobenzidene ŽDAB. with nickel chloride intensification was used as a chromogen to visualize the reaction products. The sections were then mounted onto gelatin-coated slides and coverslipped after dehydration in alcohol–xylene solutions. A Leitz Aristoplan microscope was used to obtain photomicrographs of LC and Barrington’s nucleus. 2.5. Counts of labeled nuclei In four rats per group, the regional expression of Fos-ir in Barrington’s nucleus Žleft side. was determined by counting the labeled nuclei Žone in four series of sections. in three consecutive sections. The individual counting the cells was blind to the groups.
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2.6. Statistical analysis One-way ANOVA was used for data analysis. Post-hoc analysis with Bonferroni adjustment for multiple comparisons was used to compare the experimental groups Žanimals on small and large platforms. against the dry cage control group.
3. Results 3.1. TH mRNA leÕels Rats were deprived of REM sleep for 1, 3 or 5 days by utilizing the platform method of REM sleep deprivation Žsee Section 2.. TH mRNA levels were determined after 1, 3 or 5 days on platforms. A one-way ANOVA revealed significant difference between groups in TH mRNA levels Ž F s 5.181; df s 6.46; p - 0.001.. Post-hoc analysis using Bonferroni adjustment for multiple comparisons revealed a significant increase in the levels of TH mRNA Žq32.6%; t s 4.99; df s 9; p - 0.001. in the LC of animals placed on small platform for 3 days when compared to dry cage controls ŽFig. 1A and B, Fig. 2A.. The mRNA levels remained high Žq55.4%; p - 0.003. after 5 days on small platform ŽFig. 2A.. On the other hand, the animals placed on large platform did not show any changes in TH mRNA levels compared to dry cage controls. Another set of animals were placed on small platform for 3 days and
Fig. 2. A significant increase in TH ŽA. and NET ŽB. mRNA was observed in rats placed on small platform for 3 ŽSP3. or 5 days ŽSP5.. Rats that were placed on small platform for 1 day ŽSP1. or large platform for 1 ŽLP1. or 5 days ŽLP5. did not show a significant increase in these message levels. Rats that were placed on small platforms for 3 days and then placed on large platforms for 2 days ŽSP3qLP2. also did not show any changes in TH or NET mRNA. Asterisk Ž). denotes significance compared to dry cage ŽDC. control rats.
Fig. 1. Autoradiograph depicting changes in the levels of TH and NET mRNA in the locus coeruleus. Coronal sections show that TH labeling in LC of rats deprived of REM sleep for 3 days ŽSP3. is increased ŽB. when compared to rats that were not REM sleep deprived Žthe dry cage controls, DC. ŽA.. Similarly the NET mRNA levels also increased with 3 days of REM sleep deprivation ŽSP3. animals ŽD. when compared to dry cage controls ŽC..
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transferred to large platform for 2 days. This was done in order to determine whether the changes in the TH mRNA levels in these animals were due to the REM sleep deprivation induced by the small platform and not due to the platform placement itself. In these animals the TH mRNA levels returned to baseline levels Ždry cage. indicating that when animals are placed on large platform they are able to obtain REM sleep ŽFig. 2A.. 3.2. NET mRNA leÕels The changes in the NET mRNA levels were significant as assessed by overall analysis of variance Ž F s 4.29; df s 6.14; p - 0.012.. Post-hoc analysis showed that animals placed on small platform for 3 days had a significant increase in NET mRNA Žq20.8%; t s 5.25; df s 4; p 0.006. when compared to dry cage controls ŽFig. 1C and D, Fig. 2B.. Rats placed on small platform for 5 days continued to show an increase Žq17.9%; t s 7.892; df s 4; p - 0.001. in NET mRNA levels ŽFig. 2B.. The levels of NET mRNA returned to baseline values when the animals
Fig. 4. The number of c-Fos-positive neurons in the Barrington’s nucleus were significantly higher Ž p- 0.05. after one day on both large and small platform animals when compared to the dry cage controls. After 5 days on platforms Fos immunoreactivity declined to control values. Four rats for each group were used.
were transferred to large platform for 2 days after being on small platform for 3 days ŽFig. 2B.. 3.3. Fos-ir in LC and Barrington’s nucleus The animals placed on either large or small platforms for 1 day showed increased numbers of c-Fos-ir neurons in Barrington’s nucleus. However with prolonged placement on the platforms few, if any, c-Fos-ir cells were found ŽFigs. 3 and 4.. Interestingly, in LC, c-Fos-ir neurons were not detected as a result of the REM sleep deprivation.
4. Discussion
Fig. 3. Photomicrographs of c-Fos-immunoreactive Žc-Fos-ir. neurons in the Barrington’s nucleus Žarrow.. c-Fos-ir neurons were evident in the nucleus of animals placed on small platform for one day Žphoto B. whereas both dry cage controls ŽA. and SP5 animals ŽC. did not show any c-Fos immunoreactivity.
This study examined TH and NET mRNA levels in the LC of rats deprived of REM sleep. The platform method was used to deprive the animals of REM sleep. The efficiency of the platform paradigm in inducing REM sleep deprivation was tested in our laboratory by determining the EEG and rebound sleep pattern after 1, 3 or 5 days on platforms w31x. Rats placed on small platforms but not on large platforms for 3 or 5 days showed an increased REM sleep rebound. This indicates that animals placed on small platforms but not on large platforms are deprived of REM sleep. The weights of the animals did not show any significant changes after 1, 3 or 5 days on platforms Ždata not shown.. Mendelson et al. w23x have shown that animals placed on small platforms for 4 days had a significant increase in REM sleep during the first 24 h of the rebound period. Similar increases in REM sleep have been reported after 2 days w24x or one day w30x on small platform. The TH and NET mRNA levels increased significantly after 72 h on small platform when compared to large platform and dry cage animals. These levels declined to
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control values when the animals were transferred to large platform for two days. Our results that TH mRNA levels are increased after REM sleep deprivation are in agreement with a previous report w28x describing similar effects after 72 h on small platform. In that study the animals were transferred to home cage for 24 h which resulted in a decrease of TH mRNA to control levels. In the present study, the increased TH and also NET mRNAs returned to baseline levels when the animals were transferred to LP after 72 h on small platform. These results indicate that increases in TH and NET mRNA levels are due to REM deprivation occurred due to the placement of rats on small platform. The changes in TH and NET mRNA might be due to the stress associated with the platform method of REM sleep deprivation. Increases in TH protein and mRNA have been reported to occur in response to several stress paradigms such as chronic heat exposure w7x, social stress w42x, isolation stress w1x or immobilization stress w20x. Stress mediated induction of corticotropin-releasing factor ŽCRF. increases neuronal activity in LC neurons. Such an activation is shown to result in increased c-fos mRNA expression w10x. Another group of neurons lying ventromedial to the LC known as Barrington’s nucleus, the micturition center, is also known to respond to CRF induction w40x. Both acute and chronic stress induce c-fos in Barrington’s nucleus, supporting the notion that this nucleus responds to stress w10,11x. It can be argued that animals placed on platforms Žeither on large or small. for one day experience acute stress while animals on the platforms for 3–5 days experience chronic stress. Previously it was shown that the serum corticosterone levels are high in animals placed on small and large platform for one day w28x. Our results show that c-Fos-ir in the Barrington’s nucleus was higher after one day on both small and large platform when compared to the dry cage controls and animals placed on platforms Žboth large and small. for 5 days. Collectively, these effects indicate that the animals experience stress during the first 24 h on the platform. However, the levels of TH and NET mRNA do not show any increase after one day on platforms. On the other hand, prolonged placement of animals on platforms is less likely to be stressful as they adapt to the new environment. Corticosterone levels have been shown to return to baseline levels after 72 h on large and small platform w28x. In the present study the number of c-Fos-ir neurons decreased in Barrington’s nucleus of animals placed on platforms Žeither small or large. for 5 days. The REM sleep rebound is increased only in animals placed on small platform for 5 days w31x. This suggests that the increases in TH and NET mRNA is not due to stress but is due to REM sleep deprivation. Interestingly, c-Fos-ir was not seen in LC at any tested time points. These findings may be clinically relevant to major depressive disorder. REM sleep deprivation has been shown
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to alleviate major depression w21,41,43x and we have suggested that the treatment acts by increasing the availability of monoamines at brain synapses w22x. Brain noradrenergic mechanisms are hypothesized to contribute to major depressive disorder both in humans w45x and in rat models of depression w25,44x. Reduction in norepinephrine ŽNE. levels have been suggested to result in major depression w5,13,32,34x. Tricyclic antidepressants and monoamine oxidase inhibitors increase availability of NE at the synapse and are used to treat depression. It is conceivable that the decrease in NE activity might also be associated with changes in norepinephrine transporter. In fact, recently it was shown that NET in the locus coeruleus ŽLC. is reduced in humans with major depression w14x. Our results show that REM sleep deprivation results in increased NET and TH mRNA levels. The antidepressant, desipramine, has been shown to increase NET mRNA in the LC w37,39x. This indicates that REM sleep deprivation and antidepressant treatments cause similar molecular changes in the NE system. The antidepressant effect of REM sleep deprivation might be due to increased activity of the LC neurons resulting in enhanced synthesis and release of NE at the synapse which in turn results in increased expression of the transporter. Collectively, these reports indicate that an upregulation of NET expression may contribute to alleviating depression.
Acknowledgements We would like to thank Katie Ryan and Ann-Marie Siegel for counting Fos-ir cells. This research was supported by VA Medical Research Service and NIH-NS 30140. Part of this work was presented at the 1996 annual meeting for Society for Neuroscience.
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