amyotrophic lateral sclerosis ( als ) is a progressive and fatal neurodegenerative disease characterized by the loss of lower and upper motor neurons , leading to muscle atrophy , paralysis , and death .
sleep disorders in patients with als are well - documented , and sleep - related complaints , such as insomnia , disturbed sleep , nightmare , and daytime sleepiness , have been frequently reported . moreover , several clinical studies on sleep disturbances in patients with als have been published , focusing on the frequency , characteristics , and severity of sleep problems . however , there are no animal or mechanistic studies on sleep disturbances in als
. animal and mechanistic studies on sleep disturbances in other neurodegenerative diseases , such as alzheimer 's disease ( ad ) and parkinson disease ( pd ) , might give us insights into sleep disturbances in als .
sleep problems in ad and pd involve disturbances in the neurotransmitter and hormone signaling , abnormal accumulations of neurotoxic proteins , and damage in the brain regions controlling the sleep / wake cycles , which could exist in als as well .
precursor peptide prepro - orexin , which is produced in the hypothalamic neurons , matures into two peptides , orexin a and orexin b. these peptides promote wakefulness by activating wake - active neurons ( wan ) in the hypothalamus and brain stem .
the actions of orexins are mediated by two receptors , orexin-1 ( ox1r ) and orexin-2 ( ox2r ) receptors .
we hypothesized that there are disturbances of sleep and wakefulness in the als mouse models and that orexin is an important molecule responsible for those disturbances . in the present study with sod1-g93a transgenic mice , which are extensively used as animal model for mechanistic and therapeutic studies on als
, we used sleep / wake activity recordings and molecular techniques to test our hypothesis .
transgenic sod1-g93a mice used in this study were bred from male hemizygous sod1-g93 a mice ( b6sjl - tg [ sod1-g93a ] 1 gur / j ) to female b6sjl / f1 hybrids .
the genotyping of sod1-g93a mice was performed by polymerase chain reaction ( pcr ) , as previously reported .
male hemizygous sod1-g93a mice and female b6sjl / f1 hybrids were both purchased from the jackson laboratories ( bar harbor , me , usa ) .
all mice were housed under a controlled temperature ( 22  1c ) and 12 hours : 12 hours light - dark cycle .
all animal studies were approved by the institutional animal care and use committee of peking university third hospital , and conducted in accordance with the guide for the care and use of laboratory animals of peking university . to monitor disease progression
, all mice were tested using the rota rod test apparatus ( ugo basile , varese , italy ) . the day on which a mouse first dropped off the rota rod within 600 seconds
was designated as a day of disease onset for that mouse . using this testing criterion
, we observed that all the sod1-g93a transgenic mice in this study had disease onset between 90 and 120 days of age .
thus , we determined the two - time points in our study : 90-day representing the age before disease onset and 120-day representing the age after disease onset .
twenty - eight mice were divided into four groups : sod1-g93a group at 90-day ( n = 8) , control group at 90-day ( n = 6 ) , sod1-g93a group at 120-day ( n = 6 ) , and control group at 120-day ( n = 8) . all mice received electroencephalogram / electromyogram ( eeg / emg ) recordings at 90 or 120 days of age . after performing the recordings , the cerebrospinal fluid ( csf ) and brain samples were collected .
the hypothalamus and brain stem were isolated from the brain tissue for real - time reverse transcriptase ( rt)-pcr , western blotting , and enzyme - linked immunosorbent assay ( elisa ) .
surgeries and eeg / emg recordings were performed as previously described . briefly , after anesthesia , 28 mice were implanted with eeg and emg electrodes .
the eeg electrodes were placed epidurally on the cortex , and the emg electrodes were placed in the dorsal neck muscles .
ten days after surgery , the electrodes were connected to recording cables attached to the mp150 system ( biopac , goleta , ca , usa ) .
eeg and emg recordings were performed in this manner from the freely behaving mice for 24 hours .
data were analyzed using sleep sign 2.0 software ( biopac , goleta , ca , usa ) .
total rna from the hypothalamus and brainstem tissues was extracted using trizol reagent ( takara , dalian , china ) according to the manufacturer 's instructions .
first - strand cdna was synthesized at 42c with fastquant rt kit ( tiangen , beijing , china ) using 1 g of total rna .
the amplification was performed using a superreal premix plus kit ( tiangen ) and an abi 7500 rt - pcr system ( applied biosystems , foster city , usa ) . the cdna was amplified with an initial denaturation step ( 95c , 15 minutes ) , and then with 40 pcr cycles consisting of a denaturation step ( 95c , 10 seconds ) and an annealing / extension step ( 58c , 32 seconds ) .
-actin was used as internal control to calculate the relative abundance of each mrna ( n = 46/group ) .
the specific sets of primers were as follows : prepro - orexin : f : tgaactttccttctacaaaggttc , r : caacagttcgtagagacggca ; orexin1 receptor : f : cgccaaccctatcatctacaa , r : gctctgcaaggacaaggactt ; orexin2 receptor : f : gctcaccagcataagcacact , r : tatctctttgagcagacatggg ; -actin : f : cctagcaccatgaagatcaagat , r : actcatcgtactcctgcttgct .
total protein was extracted from the hypothalamus samples using the total protein extraction kit ( applygen , beijing , china ) , consisting of radioimmunoprecipitation assay lysis buffer , phenylmethylsulfonyl fluoride , protease inhibitors , and phosphatase inhibitors , according to the manufacturer 's instructions .
protein concentration was detected by the bicinchoninic acid protein assay using a protein assay kit ( applygen ) .
samples containing equal amounts of total protein ( 20 g ) were separated by 12% sds - page and transferred to a polyvinylidene fluoride membrane ( millipore , billerica , ma , usa ) .
the membrane was blocked with 3% bovine serum albumin ( bsa ) in tris buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) for one hour , and then incubated overnight at 4c with the primary orexin antibody ( anti - orexin - prepro , 1:500 , millipore , billerica , ma , usa ) or primary -actin antibody ( anti--actin , 1:5000 , earthox , san francisco , ca , usa ) .
-actin was used as an internal loading control . the membrane was then incubated with secondary irdye 800cw goat anti - rabbit ( 1:10,000 , li - cor , lincoln , ne , usa ) or goat anti - mouse ( 1:10,000 , li - cor , lincoln , ne , usa ) antibodies at 37c for one hour .
quantitation of immunoreactive bands was performed using an odyssey infrared imaging system ( li - cor , lincoln ) .
levels of orexin a and orexin b in mouse csf and brain tissues were measured by orexin a / orexin b elisa kits ( bluegene biotech , shanghai , china ) .
mol / l , ph 7.07.2 ) per 0.5 g tissue and centrifuged . samples ( csf or brain homogenates supernatant ) or standards
were added to 96-well plates coated with anti - mouse orexin a / b antibody and elisa was performed according to manufacturer 's protocol .
the independent - samples t - test and pearson correlation analyses were performed using spss 19.0 ( spss inc . ,
transgenic sod1-g93a mice used in this study were bred from male hemizygous sod1-g93 a mice ( b6sjl - tg [ sod1-g93a ] 1 gur / j ) to female b6sjl / f1 hybrids .
the genotyping of sod1-g93a mice was performed by polymerase chain reaction ( pcr ) , as previously reported .
male hemizygous sod1-g93a mice and female b6sjl / f1 hybrids were both purchased from the jackson laboratories ( bar harbor , me , usa ) .
all mice were housed under a controlled temperature ( 22  1c ) and 12 hours : 12 hours light - dark cycle .
all animal studies were approved by the institutional animal care and use committee of peking university third hospital , and conducted in accordance with the guide for the care and use of laboratory animals of peking university .
to monitor disease progression , all mice were tested using the rota rod test apparatus ( ugo basile , varese , italy ) . the day on which a mouse first dropped off the rota rod within 600 seconds
was designated as a day of disease onset for that mouse . using this testing criterion
, we observed that all the sod1-g93a transgenic mice in this study had disease onset between 90 and 120 days of age .
thus , we determined the two - time points in our study : 90-day representing the age before disease onset and 120-day representing the age after disease onset .
twenty - eight mice were divided into four groups : sod1-g93a group at 90-day ( n = 8) , control group at 90-day ( n = 6 ) , sod1-g93a group at 120-day ( n = 6 ) , and control group at 120-day ( n = 8) . all mice received electroencephalogram / electromyogram ( eeg / emg ) recordings at 90 or 120 days of age . after performing the recordings , the cerebrospinal fluid ( csf ) and brain samples were collected .
the hypothalamus and brain stem were isolated from the brain tissue for real - time reverse transcriptase ( rt)-pcr , western blotting , and enzyme - linked immunosorbent assay ( elisa ) .
surgeries and eeg / emg recordings were performed as previously described . briefly , after anesthesia , 28 mice were implanted with eeg and emg electrodes .
the eeg electrodes were placed epidurally on the cortex , and the emg electrodes were placed in the dorsal neck muscles .
ten days after surgery , the electrodes were connected to recording cables attached to the mp150 system ( biopac , goleta , ca , usa ) .
eeg and emg recordings were performed in this manner from the freely behaving mice for 24 hours .
data were analyzed using sleep sign 2.0 software ( biopac , goleta , ca , usa ) .
total rna from the hypothalamus and brainstem tissues was extracted using trizol reagent ( takara , dalian , china ) according to the manufacturer 's instructions .
first - strand cdna was synthesized at 42c with fastquant rt kit ( tiangen , beijing , china ) using 1 g of total rna .
the amplification was performed using a superreal premix plus kit ( tiangen ) and an abi 7500 rt - pcr system ( applied biosystems , foster city , usa ) . the cdna was amplified with an initial denaturation step ( 95c , 15 minutes ) , and then with 40 pcr cycles consisting of a denaturation step ( 95c , 10 seconds ) and an annealing / extension step ( 58c , 32 seconds ) .
-actin was used as internal control to calculate the relative abundance of each mrna ( n = 46/group ) .
the specific sets of primers were as follows : prepro - orexin : f : tgaactttccttctacaaaggttc , r : caacagttcgtagagacggca ; orexin1 receptor : f : cgccaaccctatcatctacaa , r : gctctgcaaggacaaggactt ; orexin2 receptor : f : gctcaccagcataagcacact , r : tatctctttgagcagacatggg ; -actin : f : cctagcaccatgaagatcaagat , r : actcatcgtactcctgcttgct .
total protein was extracted from the hypothalamus samples using the total protein extraction kit ( applygen , beijing , china ) , consisting of radioimmunoprecipitation assay lysis buffer , phenylmethylsulfonyl fluoride , protease inhibitors , and phosphatase inhibitors , according to the manufacturer 's instructions .
protein concentration was detected by the bicinchoninic acid protein assay using a protein assay kit ( applygen ) .
samples containing equal amounts of total protein ( 20 g ) were separated by 12% sds - page and transferred to a polyvinylidene fluoride membrane ( millipore , billerica , ma , usa ) .
the membrane was blocked with 3% bovine serum albumin ( bsa ) in tris buffered saline ( tbs ) containing 0.1% tween 20 ( tbs - t ) for one hour , and then incubated overnight at 4c with the primary orexin antibody ( anti - orexin - prepro , 1:500 , millipore , billerica , ma , usa ) or primary -actin antibody ( anti--actin , 1:5000 , earthox , san francisco , ca , usa ) .
the membrane was then incubated with secondary irdye 800cw goat anti - rabbit ( 1:10,000 , li - cor , lincoln , ne , usa ) or goat anti - mouse ( 1:10,000 , li - cor , lincoln , ne , usa ) antibodies at 37c for one hour .
quantitation of immunoreactive bands was performed using an odyssey infrared imaging system ( li - cor , lincoln ) .
levels of orexin a and orexin b in mouse csf and brain tissues were measured by orexin a / orexin b elisa kits ( bluegene biotech , shanghai , china ) .
brain tissues were homogenized in 1 ml pbs ( 0.02 mol / l , ph 7.07.2 ) per 0.5 g tissue and centrifuged . samples ( csf or brain homogenates supernatant ) or standards were added to 96-well plates coated with anti - mouse orexin a / b antibody and elisa was performed according to manufacturer 's protocol .
the independent - samples t - test and pearson correlation analyses were performed using spss 19.0 ( spss inc . ,
sleep / wake recordings were performed in both sod1-g93a transgenic mice and their control groups . in the 90-day sod1-g93a transgenic mice , across a 24-hour recording period , the total sleep time ( tst ) was significantly decreased ( [ 443.23  40.42 minutes ] vs. [ 569.97  39.04 minutes ] , p < 0.05 ) and wakefulness was increased ( [ 996.78  40.42 minutes ] vs. [ 870.03  39.04 minutes ] , p < 0.05 ) , compared to the littermate controls .
non - rapid eye movement ( nrem ) sleep ( [ 411.29  35.41 minutes ] vs. [ 533.48  37.01 minutes ] , p < 0.05 ) and deep sleep ( ds ) ( [ 47.35  9.55 minutes ] vs. [ 107.35  11.36 minutes ] , p < 0.01 ) were also reduced .
no significant difference was found in rem sleep and light sleep between the two groups for the 24-hour period [ figure 1a ] .
eeg / emg recordings for 24 hours from the sod1-g93a transgenic mice and littermate control mice at 90 days and 120 days of age .
( a ) tst , nrem , and ds are significantly decreased in the 90-day sod1-g93a transgenic mice .
( b ) in the 120-day sod1-g93a transgenic mice , wake is also significantly enhanced .
tst : total sleep time ; wake : wakefulness ; rem : rapid eye movement sleep ; nrem : non - rapid eye movement sleep ; ls : light sleep ; ds : deep sleep ; eeg / emg : electroencephalogram / electromyogram . in the 120-day sod1-g93a transgenic mice ,
a remarkable increase in wakefulness ( [ 1110.33  38.16 minutes ] vs. [ 804.29  74.57 minutes ] , p < 0.01 ) , and a decrease in tst ( [ 329.67  38.16 minutes ] vs. [ 635.71  74.57 minutes ] , p < 0.01 ) , nrem ( [ 306.03  36.81 minutes ] vs. [ 534.68  82.68 minutes ] , p < 0.05 ) and ds ( [ 22.88  10.27 minutes ] vs. [ 107.90  25.21 minutes ] , p < 0.05 ) were observed . however , in contrast with the 90-day sod1-g93a transgenic mice , rem sleep was decreased ( [ 23.62  9.96 minutes ] vs. [ 101.08  13.93 minutes ] , p < 0.01 ) in the 120-day sod1-g93a transgenic mice compared to the 120-day littermate controls [ figure 1b ] .
next , we evaluated the changes in the orexin system in the sod1-g93a transgenic mice .
first , we tested the level of mrna and protein expression of prepro - orexin .
q - pcr showed increased levels of prepro - orexin mrna in the hypothalamus of the 90-day ( 1.84  0.24 vs. 1.00  0.20 , p < 0.05 ) and 120-day ( 2.06  0.25 vs. 1.00  0.14 , p < 0.01 ) sod1-g93a transgenic mice , compared to controls [ figure 2a ] .
the western blotting analysis also showed increased levels of prepro - orexin protein in the hypothalamus of both 90-day ( 2.33  0.24 vs. 1.00  0.18 , p < 0.01 ) and 120-day ( 2.30  0.14 vs. 1.00  0.19 , p < 0.01 ) sod1-g93a transgenic mice as compared to littermate controls [ figure 2b ] . prepro - orexin increases in sod1-g93a transgenic mice .
( a ) q - pcr was performed in the hypothalamic tissues from the sod1-g93a transgenic mice and control groups .
prepro - orexin mrna is significantly elevated in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice , compared to control .
( b ) western blotting was performed with antibody anti - orexin - prepro in the sod1-g93a transgenic mice and control groups .
prepro - orexin expression is increased in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice as compared to control .
data are expressed as mean  standard error of mean ( n = 46/group ) .
we then tested the protein levels of orexin a and b , using elisa . in the hypothalamus , the level of orexin a was significantly enhanced in the 90-day ( [ 2126.47  70.65 pg / mg ] vs. [ 1591.52  61.25
pg / mg ] protein , p < 0.01 ) and 120-day ( [ 2166.32  115.98
pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice , compared to their respective littermate control mice [ figure 3a ] .
the level of orexin a was also elevated significantly in the brain stem of the 90-day ( [ 1540.93  34.87 pg / mg ] vs. [ 1170.04  47.73
pg / mg ] protein , p < 0.01 ) and 120-day ( [ 1583.96  21.64 pg / mg ] vs. [ 1224.87  51.62 pg / mg ] protein , p
there was no significant difference in orexin a levels between the sod1-g93a transgenic mice and control groups in the csf , at either time points [ figure 3c ] .
similarly , orexin b levels were also significantly increased in the hypothalamus of the 90-day ( [ 3485.35  74.62 pg / mg ] vs. [ 2352.45  142.56
pg / mg ] protein , p < 0.01 ) and 120-day ( [ 3656.34  139.17
 117.70 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice [ figure 3d ] . in the brain stem , orexin b was also significantly enhanced in both transgenic groups ( 90-day : [ 2726.79  34.06 pg / mg ] vs. [ 2411.70  64.18 pg / mg ] protein , p < 0.01 and 120-day : [ 2736.45  57.32 pg / mg ] vs. [ 2369.33  83.57 pg / mg ] protein , p < 0.05 ) as compared to littermate controls [ figure 3e ] . in the csf ,
no significant difference was found in orexin b levels between the respective transgenic and control groups [ figure 3f ] . orexin a and b increase in sod1-g93a transgenic mice .
( a - c ) elisa reveals that orexin a levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice than controls , but there is no significant difference for orexin a in the csf .
( d - f ) elisa shows increased the level of orexin b in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice compared with control , but no significant difference is seen in the csf .
ht : hypothalamus ; bs : brain stem ; csf : cerebrospinal fluid ; elisa : enzyme - linked immunosorbent assay .
q - pcr analysis revealed no significant difference in the mrna levels of ox1r [ figure 4a and 4b ] and ox2r [ figure 4c and 4d ] in the hypothalamus or the brain stem between the sod1-g93a transgenic mice and controls .
q - pcr of orexin-1 receptor mrna show no significant difference in the hypothalamus ( a ) and brain stem ( b ) of the 90 and 120 days sod1-g93a transgenic mice than controls .
no significant difference is found in orexin-2 receptor mrna in the hypothalamus ( c ) and brain stem ( d ) of the 90 and 120 days sod1-g93a transgenic mice than controls .
pearson correlation analyses were performed between sleep / wake time ( tst , wake , rem , nrem , and ds ) and expression levels of orexins ( prepro - orexin , orexin a and b ) .
absolute values of the correlation coefficient confirmed high correlation between the sleep / wake time ( tst , wake , and nrem ) and expression levels of orexins .
meanwhile , there was a moderate correlation between rem / ds and orexins [ table 1 ] .
pearson correlation coefficient of sleep / wake time and orexins tst : total sleep time ; wake : wakefulness ; rem : rapid eyes movement sleep ; nrem : nonrapid eyes movement sleep ; ds : deep sleep ; ht : hypothalamus ; bs : brain stem .
sleep / wake recordings were performed in both sod1-g93a transgenic mice and their control groups . in the 90-day sod1-g93a transgenic mice , across a 24-hour recording period , the total sleep time ( tst ) was significantly decreased ( [ 443.23  40.42 minutes ] vs. [ 569.97  39.04 minutes ] , p < 0.05 ) and wakefulness was increased ( [ 996.78  40.42 minutes ] vs. [ 870.03  39.04 minutes ] , p < 0.05 ) , compared to the littermate controls .
non - rapid eye movement ( nrem ) sleep ( [ 411.29  35.41 minutes ] vs. [ 533.48  37.01 minutes ] , p < 0.05 ) and deep sleep ( ds ) ( [ 47.35  9.55 minutes ] vs. [ 107.35  11.36 minutes ] , p < 0.01 ) were also reduced .
no significant difference was found in rem sleep and light sleep between the two groups for the 24-hour period [ figure 1a ] .
eeg / emg recordings for 24 hours from the sod1-g93a transgenic mice and littermate control mice at 90 days and 120 days of age .
( a ) tst , nrem , and ds are significantly decreased in the 90-day sod1-g93a transgenic mice .
( b ) in the 120-day sod1-g93a transgenic mice , wake is also significantly enhanced .
tst : total sleep time ; wake : wakefulness ; rem : rapid eye movement sleep ; nrem : non - rapid eye movement sleep ; ls : light sleep ; ds : deep sleep ; eeg / emg : electroencephalogram / electromyogram . in the 120-day sod1-g93a transgenic mice ,
a remarkable increase in wakefulness ( [ 1110.33  38.16 minutes ] vs. [ 804.29  74.57 minutes ] , p < 0.01 ) , and a decrease in tst ( [ 329.67  38.16 minutes ] vs. [ 635.71  74.57 minutes ] , p < 0.01 ) , nrem ( [ 306.03  36.81 minutes ] vs. [ 534.68  82.68 minutes ] , p < 0.05 ) and ds ( [ 22.88  10.27 minutes ] vs. [ 107.90  25.21 minutes ] , p < 0.05 ) were observed . however , in contrast with the 90-day sod1-g93a transgenic mice , rem sleep was decreased ( [ 23.62  9.96 minutes ] vs. [ 101.08  13.93 minutes ] , p < 0.01 ) in the 120-day sod1-g93a transgenic mice compared to the 120-day littermate controls [ figure 1b ] .
next , we evaluated the changes in the orexin system in the sod1-g93a transgenic mice .
first , we tested the level of mrna and protein expression of prepro - orexin .
q - pcr showed increased levels of prepro - orexin mrna in the hypothalamus of the 90-day ( 1.84  0.24 vs. 1.00  0.20 , p < 0.05 ) and 120-day ( 2.06  0.25 vs. 1.00  0.14 , p < 0.01 ) sod1-g93a transgenic mice , compared to controls [ figure 2a ] .
the western blotting analysis also showed increased levels of prepro - orexin protein in the hypothalamus of both 90-day ( 2.33  0.24 vs. 1.00  0.18 , p < 0.01 ) and 120-day ( 2.30  0.14 vs. 1.00  0.19 , p < 0.01 ) sod1-g93a transgenic mice as compared to littermate controls [ figure 2b ] .
( a ) q - pcr was performed in the hypothalamic tissues from the sod1-g93a transgenic mice and control groups .
prepro - orexin mrna is significantly elevated in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice , compared to control .
( b ) western blotting was performed with antibody anti - orexin - prepro in the sod1-g93a transgenic mice and control groups .
prepro - orexin expression is increased in the hypothalamus of the 90 and 120 days sod1-g93a transgenic mice as compared to control .
data are expressed as mean  standard error of mean ( n = 46/group ) .
we then tested the protein levels of orexin a and b , using elisa . in the hypothalamus , the level of orexin a was significantly enhanced in the 90-day ( [ 2126.47  70.65 pg / mg ] vs. [ 1591.52  61.25
pg / mg ] protein , p < 0.01 ) and 120-day ( [ 2166.32  115.98 pg / mg ] vs. [ 1446.18  41.31 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice , compared to their respective littermate control mice [ figure 3a ] .
the level of orexin a was also elevated significantly in the brain stem of the 90-day ( [ 1540.93  34.87 pg / mg ] vs. [ 1170.04  47.73
pg / mg ] protein , p < 0.01 ) and 120-day ( [ 1583.96  21.64 pg / mg ] vs. [ 1224.87  51.62 pg / mg ] protein , p < 0.01 ) transgenic groups [ figure 3b ] .
there was no significant difference in orexin a levels between the sod1-g93a transgenic mice and control groups in the csf , at either time points [ figure 3c ] .
similarly , orexin b levels were also significantly increased in the hypothalamus of the 90-day ( [ 3485.35  74.62 pg / mg ] vs. [ 2352.45  142.56
pg / mg ] protein , p < 0.01 ) and 120-day ( [ 3656.34  139.17 pg / mg ] vs. [ 2411.57  117.70 pg / mg ] protein , p < 0.01 ) sod1-g93a transgenic mice [ figure 3d ] . in the brain stem , orexin b was also significantly enhanced in both transgenic groups ( 90-day : [ 2726.79  34.06
 64.18 pg / mg ] protein , p < 0.01 and 120-day : [ 2736.45  57.32 pg / mg ] vs. [ 2369.33  83.57 pg / mg ] protein , p < 0.05 ) as compared to littermate controls [ figure 3e ] . in the csf , no significant difference was found in orexin b levels between the respective transgenic and control groups [ figure 3f ] . orexin a and b increase in sod1-g93a transgenic mice .
( a - c ) elisa reveals that orexin a levels are enhanced in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice than controls , but there is no significant difference for orexin a in the csf .
( d - f ) elisa shows increased the level of orexin b in the hypothalamus and brain stem of the 90 and 120 days sod1-g93a transgenic mice compared with control , but no significant difference is seen in the csf .
ht : hypothalamus ; bs : brain stem ; csf : cerebrospinal fluid ; elisa : enzyme - linked immunosorbent assay .
q - pcr analysis revealed no significant difference in the mrna levels of ox1r [ figure 4a and 4b ] and ox2r [ figure 4c and 4d ] in the hypothalamus or the brain stem between the sod1-g93a transgenic mice and controls .
q - pcr of orexin-1 receptor mrna show no significant difference in the hypothalamus ( a ) and brain stem ( b ) of the 90 and 120 days sod1-g93a transgenic mice than controls .
no significant difference is found in orexin-2 receptor mrna in the hypothalamus ( c ) and brain stem ( d ) of the 90 and 120 days sod1-g93a transgenic mice than controls .
pearson correlation analyses were performed between sleep / wake time ( tst , wake , rem , nrem , and ds ) and expression levels of orexins ( prepro - orexin , orexin a and b ) .
absolute values of the correlation coefficient confirmed high correlation between the sleep / wake time ( tst , wake , and nrem ) and expression levels of orexins .
meanwhile , there was a moderate correlation between rem / ds and orexins [ table 1 ] .
pearson correlation coefficient of sleep / wake time and orexins tst : total sleep time ; wake : wakefulness ; rem : rapid eyes movement sleep ; nrem : nonrapid eyes movement sleep ; ds : deep sleep ; ht : hypothalamus ; bs : brain stem .
in summary , we observed marked sleep disturbances in the sod1-g93a mouse model of als , and demonstrated that the increase in expression of orexins correlated with these sleep disturbances in these mice .
sleep / wake disturbances are often reported by patients with als , but no related animal studies have been published .
the exact mechanism behind the sleep / wake disturbances in als remains to be determined . in the present study , for the first time , we observed enhanced wakefulness and reduced sleep time ( including tst , nrem , and ds ) in both 90-day and 120-day sod1-g93a transgenic mice .
these results indicate that sleep / wake disturbances are early symptoms in these sod1-g93a transgenic mice , and these disturbances may be correlated with the onset of als . in addition , rem sleep time was significantly decreased in the 120-day sod1-g93a transgenic mice as compared to the 90-day sod1-g93a transgenic mice , suggesting an aggravation and the possible link of sleep disturbances with disease progression .
therefore , studies on sleep disturbances in als are of significance to elucidate the etiology and pathogenesis of als . moreover , considering the early occurrence of sleep disorders in ad and pd , we speculate that the dysregulation of sleep might be common in the early stages of neurodegenerative diseases . to further investigate the mechanism of sleep / wake disturbances in als
sleep is a complicated behavior regulated by wakefulness promoting systems , sleep promoting systems , and circadian rhythms .
wakefulness is promoted by multiple groups of wan , which are mainly located in the brain stem and hypothalamus , including cholinergic neurons , orexinergic neurons , and dopaminergic neurons .
neurotransmitters in these wan , including acetylcholine , dopamine , glutamate , histamine , norepinephrine , orexin , and serotonin , promote wakefulness .
prepro - orexin , which is produced in hypothalamic neurons , matures into two neuropeptides , orexin a and orexin b. these two peptides activate monoaminergic and cholinergic neurons in the hypothalamus and brain stem to maintain a long , consolidated awake period . in our study ,
prepro - orexin was increased in the hypothalamus in the 90 and 120 days sod1-g93a transgenic mice .
orexin a and b were significantly enhanced in the hypothalamus and brain stem of the 90 days and 120 days sod1-g93a transgenic mice compared with littermate controls .
these results suggest a strong association between increased expression of orexins and the extension of wakefulness in als mice .
pearson correlation analysis , which showed moderate to high correlations between sleep / wake time and expression of orexins , further confirmed this association .
these results indicate that the extension of wakefulness and sleep / wake disturbances in the als mice may be caused , at least partly , by increase in expression of orexins . according to the absolute value of the correlation coefficient in table 1 , nrem had the highest correlation with expression of orexins among all stages of sleep .
however , no differences were detected in the levels of orexin receptors in the hypothalamus and brain stem between the als mice and control groups , suggesting that enhanced wakefulness and other sleep disturbances in these transgenic mice were not promoted by upregulation of orexin receptors .
in addition , the levels of orexin a and b were only elevated in the brain tissue other than the csf in the sod1-g93a transgenic mice .
this is consistent with the clinical study by van rooij et al . , which demonstrated that csf orexin levels were normal in patients with als .
sleep disturbances occur before disease onset in the als sod1-g93a transgenic mice . increased expression of orexins
further experiments need to be carried out in the future to investigate the underlying mechanisms .
 background : sleep / wake disturbances in patients with amyotrophic lateral sclerosis ( als ) are well - documented , however , no animal or mechanistic studies on these disturbances exist . 
 orexin is a crucial neurotransmitter in promoting wakefulness in sleep / wake regulation , and may play an important role in sleep disturbances in als . in this study 
 , we used sod1-g93a transgenic mice as an als mouse model to investigate the sleep / wake disturbances and their possible mechanisms in als.methods:electroencephalogram/electromyogram recordings were performed in sod1-g93a transgenic mice and their littermate control mice at the ages of 90 and 120 days , and the samples obtained from these groups were subjected to quantitative reverse transcriptase - polymerase chain reaction , western blotting , and enzyme - linked immunosorbent assay.results:for the first time in sod1-g93a transgenic mice , we observed significantly increased wakefulness , reduced sleep time , and up - regulated orexins ( prepro - orexin , orexin a and b ) at both 90 and 120 days . correlation analysis confirmed moderate to high correlations between sleep / wake time ( total sleep time , wakefulness time , rapid eye movement [ rem ] sleep time , non - rem sleep time , and deep sleep time ) and increase in orexins ( prepro - orexin , orexin a and b).conclusion : sleep / wake disturbances occur before disease onset in this als mouse model . 
 increased orexins may promote wakefulness and result in these disturbances before and after disease onset , thus making them potential therapeutic targets for amelioration of sleep disturbances in als . 
 further studies are required to elucidate the underlying mechanisms in the future . 
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Animals
Experimental design
Sleep-wake activity recordings
Quantitative reverse transcriptase-polymerase chain reaction
Western blotting
Enzyme-linked immunosorbent assay
Statistical analysis
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Disturbances of sleep and wakefulness in SOD1-G93A transgenic mice
Orexins increase in SOD1-G93A transgenic mice
Correlation analyses
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