Pergamon Press
Life Sciences Vol . 22, pp . 445-450 Printed in the U .S .A .
CEREBROSPINAL FLUID CYCLIC AMP LEVELS IN RHESUS DAILY FLUCTUATIONS MONKEYS : Jonathan B . Ratz, Charles Valases, George N. Catravas, Sanford Wright, Jr .* Biochemistry Department and Neurobiology Department* Armed Forces Radiobiology Research Institute Bethesda, Maryland 20014 and Paul Pevsner, Thomas Fouts Section of Neuroradiology Medical College of Virginia Richmond, Virginia (Received in final form October 14, 1977) Summary Daily fluctuations in cerebrospinal fluid cyclic AMP levels were found in neurologically intact rhesus monkeys . When animals were maintained in continuous dim light as opposed to a 12L/12D regimen, the fluctuations in cerebrospinal fluid cyclic AMP levels .appeared to be reduced. Knowledge of these daily fluctuations may be helpful for protocols of future experiments concerning measurements of CSF cAMP . Cyclic AMP (cAMP) has been suggested as a second messenger in central nervous system synaptic events, both by analogy with its role in other tissues and in consequence of in vitro. and in vivo experiments with nervous tissues (1-6) . Increases in cerebrospinal fluid (CSF) cAMP levels have resulted from in vivo administration of putative adrenergic transmitters or melanotropic and lipolytic peptides to ventricular fluid (1,2,7) . Other experimental and clinical conditions have been correlated with alterations of CSF cAMP levels including epilepsy (8), coma subsequent to head trauma (9), hydrocephalus (10), elevated intracranial pressure (10), cerebral infarction (11), transient cerebral ischemia (migraine headaches) (11), and pyrogen-induced fever (12,13) . Yet in contrast to these efforts to relate complex pathophysiological states with CSF cAMP levels, little is known about such elementary findings as the wide normal range of CSF cAMP values (14,15) . Therefore, we decided to examine neurologically intact rhesus monkeys in order to determine the presence or absence of intrinsic daily fluctuations in CSF cAlO? levels . Methods One female and two male rhesus monkeys (4 .5-6 .0 kg) were chronically implanted with Ommaya CSF reservoirs connected to silastic catheters placed in the fourth ventricle . This method permitted sterile aspirations of 0.5-1 .0 ml of CSF from the awake animal on a chronic basis . Details of this technique have been previously published (16) . Three mCi of 99mfc were injected into the reservoir followed by extensive, repeated manual compression and passive filling 445
44 6
Daily Variances in CSF cAMP Levels
Vol . 22, No . 6, 1978
of the reservoir . Radiocisternography of the ventricular system revealed CSF exchange between the ventricular system, the posterior fossa, and the upper spinal subarachnoid space . Computerized tomography revealed a neuroradiologically normal status with absence of hydrocephalus in all animals . To insure equilibration of CSF between the reservoir and the subarachnoid space, reservoirs were compressed and allowed to fill several times before sampling . Our initial studies demonstrated no significant loss in CSF cAMP content when CSF was allowed to incubate at 25 0C for 2 hours . This tends to discount possible decomposition of cAMP while in the reservoir despite reported occurrence of phosphodiesterase in CSF (17) . Animals were placed in chairs, enclosed in separate chambers, and fed regularly at 8 a .m . and 3 p .m . In the first experiences, monkeys were maintained on a 12-hour light/12-hour dark (12L/12D) lighting schedule (lighting was 20 foot-candles) . CSF samples (0 .5 ml) were taken every 3 hours over a 72-hour period . In later experiments, animals were adapted to continuous dim light (less than 0 .25 foot-candles) for 5 days before the 72-hour sampling period commenced . Aspirated samples were immediately frozen in liquid nitrogen They were later deproteinized with 0.5 ml of 6% trichloroand stored at -801C . acetic acid, ether-extracted to remove the acid, lyophilized, resuspended in buffer, and assayed radioimmunologically for cAMP content (18,19) . Radioimmunoassay kits were purchased from Schwartz-Mann Co ., Orangeburg, N . J . because intracranial CAMP may increase in stressed animals (20), the necessary restraint for and discomfort of withdrawing CSF from the Ommaya reservoirs of awake monkeys may have iatrogenically influenced the levels of cANP . Therefore, CSF samples were twice withdrawn from each of 4 monkeys both before and 1 hour after the induction of a light ketamine (10 mgm/kg) and atropine ( .004 mgm/kg) anesthesia . The preanesthesia value did not differ significantly from the 1 hour anesthetized value (Preanesthesia - 4 .9 _+ 1 .7 and anesthesia - 5 .9 _+ 2 .8 pmoles CAMP/ml CSF, respectively) . These results provide evidence that the observed fluctuations of CSF cAMP measured in this study were more likely due to factors other than a simple reflection of a stress induced to awake animals during withdrawal of CSF. Results and Discussion Part a of Figure 1 contains three records of monkey CSF cAMP content as a function of time during the 72-hour period, using animals exposed to a 12L/12D Fluctuations in cANP levels appear to be evident in-all light schedule . subjects . Considerable variability in cAMP levels in different subjects is suggested and is consistent with data on the wide range of cAMP levels in human CSF (14,15) . Part b of Figure 1 demonstrates that when the animals were maintained in continuous dim light for 5 days prior to the 72-hour dim light sampling period, a trend is evident toward a lower mean CAMP level in the dim light condition (18.9 pmoles cAMP/ml CSF in 12L/12D animals versus 10 .8 pmoles/ml in dim light animals) . This interpretation would be consistent with recent work on neural cyclic nucleotide rhythms influenced by lighting schedules (21-23) . In order to examine the variability of the cAMP measurements themselves, the coefficients of variation (S .D ./average) (24) of 9 pairs of calibration measurements from each of three different radioimmunoassay kits were averaged . The pooled coefficient was 0 .25 . One can obtain an estimate of the standard deviation at any cAMP concentration by multiplying 0 .25 by the concentration in pmoles of CAMP/ml CSF . For example, at 10 pmoles CAMP/ml CSF an estimate of the standard deviation (or measurement error) would be 2 .5 pmoles cAMP/ml CSF.
Daily Variances in OF cANP Levels
Vol . 22, No . 6, 1978
0600 90
1900
0600
TIME (hours) 0600 1800
1800
447
0600
90 70, 40
i
s 3: t
20
Y
10 0 70
0 l/24 D
46
ûE
n Y
20
10 0 0600
1900
0600
DAY 1 --
0600 1000 TIME (boon) DAY 2
1800
0600
DAY 3
FIG . 1 Cerebrospinal fluid cyclic AMP levels in rhesus Three monkeys were monkeys over a 72-hour period . accustomed either to a 12-hour light/12-hour dark schedule (12L/12D) as presented in (a) or to a 24hour dim light schedule (0 .25 foot-candles) (OL/24D) as shown in (b) . As stated above, the average cANP levels of dim light animals was 10 .8 (± 7 .3 S .D .), whereas for the light/dark animals the average was 18 .9 (+ 17 .2 S .D .) ; n - 71 for each mean . Since the S .D . of the light/dark animals (17 .2) is greater than that of the dim-light animals (7 .3), one would tend to believe that the observed fluctuations of CSF cAMP levels were not only significant with respect to measurement error, but also more prominent under light/dark conditions . Because of the nature of these experiments with primates, the lack of a large number of measurements does not allow one to perform a valid statistical test of these apparent differences .
44 8
Daily Variances in CSF cAMP Levels
Vol . 22, No . 6, 1978
When the results from all monkeys were evaluated no statistically significant dark/light differences for the 12-hour light/12-hour dark group were found . The Fried-n two-way analysis (25) was employed for this mathe matical examination. This statistical test is a non-parametric version of the analysis, of variance . It is insensitive to wild excursions of data, removes the caveat of correlated observations and adjusts for animal to animal differences. Possibly the lack of statistical significance might only mean that the sample size was too small to detect the differences suggested by the individual profiles in Figure 1 . In the future, the number of CSF samples might be increased by either using an automated, continuous 24-hour system of CSF withdrawal and collection, or the allocation of considerably greater numbers of enclosed, individual chambers for placement of additional numbers of animals. It is not clear from our experiments whether the changes we observed in CSF cAMP levels reflect changes in brain cANP, changes in transport of cMP from brain to CSF, degradation of CAMP within the intercellular spaces of the brain, or a combination of these factors . The potential influence of environmental lighting upon CSF cAMP concentration remains to be delineated . Knowledge of temporal fluctuations of CSF cANP levels demonstrated in our study may be useful in future experiments. Acknowledgment Special thanks are extended to Dr . Sheldon Levin and Mr . William Jackson of the Biostatistics Office of the Armed Forces Radiobiology Research Institute, Bethesda, Maryland, for their help in the statistical analysis of these data . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
J. B. SEBENS and J. KORF, Exp . Neurol . 46 333-344 (1975) . J. KORF, P. H . BOER, and D . FEKKES, Brain Res . 113 551-561 (1976) . J. W. KEBABIAN and P . GREENGARD, Science 174 1346-1349 (1971) . .SEGAL, and A. P . OLIVER, Adv. F. E. BLOOM, G. R . SIGGINS, B. J. HOFFER, M Cyclic Nucleotide Res . _5 603-618 (1975) . J. W . KEBABIAN, A. L . STEINER, and P . GREENGARD, J . Pharm. Exp. Ther . 19 3 474-488 (1975) . G. C. PALMER, F. SULSER, and G. A. ROBINSON, Neuropharmacology 12 327-337 (1973) . D. RUDMAN and J. W. ISAACS, Endocrinology 97 1476-1480 (1975) . V . V . MYLLA, E . R. HEIKKINEN, H . VAPAATALO, and E. HOKKANEN, Eur . Neurol . _13 123-130 (1975) . D . RUDMAN, A . FLEISCHER, and M. H. KITNER, New Engl . J . Med. 295 635-638 (1976) . D. RUDMAN, M. S. O'BRIEN, A. S . McKINLEY, J. C . HOFFMAN, and J . H.PATTERSON, J . Clin . Endocrinology Metab . 42 1088-1097 (1976) . K. M. A. WELCH, E . CHABI, J . H. NELL, K. BARTOSH, A. N . C . CHEE, N. T. MATTHEW, and V. S . ACHAR, Headache 16 160-167 (1976) . W. K. PHILLIP-DORMSTON, Pfluegers Arch . 362 223-227 (1976) . W. K. PHILLIP-DORMSTON and R . SIEGERT, Med . Microbiol . Immunol . (Berl .) 161 11-13 (1975) . B . B . BROOKS, J . SODÉ, W. K. ENGEL, Cyclic Nucleotide Metabolism in Neuromuscular Disease, presented at UCLA Conf . on Research Trends in Amyotrophic Lateral Sclerosis, Los Angeles, Calif., Oct 18-21 (1975) . V. V. MYLLA, V. VAPAATALO, E . HOKKANEN, and E . R. HEIKKINEN, Eur . Neurol . 12 28-32 (1972) . J . H. WOOD, D . G . POPLACK, W. A. BLEYER, and A . K. OMMAYA, Science 195 499501 (1977) .
Vol . 22, No . 6, 1978
Daily Variences in CSF cAMP Levels
449
17 . H . HIKAKA, M . SHIBUYA, T . ASANO, and F . HARA, J . Neurochem . 25 49-53 (1975) . 18 . A . L . STEINER, C . W . PARKER, and D . M . KIPNIS, J . Biol . Chem . 247 11061113 (1972) . 19 . A . L . STEINER, A . S . PAGLIARI, L . R . CHASE, and D . M .KKIPNIS, J . Biol . Chem. 274 1114-1120 (1972) . 20 .'i.L . DELAPAZ, S . R . DICKMAN, and B . I . GROSSER, Brain Research 85 171-175 (1975) . 21 . K . P . MINNEMAN and L . L . IVERSEN, Nature 260 59-61 (1976) . 22 . J . A . ROMERO, Fed . Proc . 35 1157-1161 (1976) . 23 . M . PERLO, B . FESTHOFF, M . EBERT, E . K . GORDON, M . G . ZIEGLER, C . R . LAKE, H . HOFFMAN, D . K . JOHNSON, and T . N . CHASE, Neuroscience Abstr . _2 499Soc . Neurosc ., Bethesda, Md . (1976) . 24 . R . G . D . STEE L and J . H . TORRIE, Principles and Procedures of Statistics , p . 20, McGraw-Hill (1960) . 25 . W . J . CONOVER, Practical Nonparametric Statistics p . 224, Wiley and Sons (1971) .
Life Sciences Vol . 22, pp . 445-450 Printed in the U .S .A .
CEREBROSPINAL FLUID CYCLIC AMP LEVELS IN RHESUS DAILY FLUCTUATIONS MONKEYS : Jonathan B . Ratz, Charles Valases, George N. Catravas, Sanford Wright, Jr .* Biochemistry Department and Neurobiology Department* Armed Forces Radiobiology Research Institute Bethesda, Maryland 20014 and Paul Pevsner, Thomas Fouts Section of Neuroradiology Medical College of Virginia Richmond, Virginia (Received in final form October 14, 1977) Summary Daily fluctuations in cerebrospinal fluid cyclic AMP levels were found in neurologically intact rhesus monkeys . When animals were maintained in continuous dim light as opposed to a 12L/12D regimen, the fluctuations in cerebrospinal fluid cyclic AMP levels .appeared to be reduced. Knowledge of these daily fluctuations may be helpful for protocols of future experiments concerning measurements of CSF cAMP . Cyclic AMP (cAMP) has been suggested as a second messenger in central nervous system synaptic events, both by analogy with its role in other tissues and in consequence of in vitro. and in vivo experiments with nervous tissues (1-6) . Increases in cerebrospinal fluid (CSF) cAMP levels have resulted from in vivo administration of putative adrenergic transmitters or melanotropic and lipolytic peptides to ventricular fluid (1,2,7) . Other experimental and clinical conditions have been correlated with alterations of CSF cAMP levels including epilepsy (8), coma subsequent to head trauma (9), hydrocephalus (10), elevated intracranial pressure (10), cerebral infarction (11), transient cerebral ischemia (migraine headaches) (11), and pyrogen-induced fever (12,13) . Yet in contrast to these efforts to relate complex pathophysiological states with CSF cAMP levels, little is known about such elementary findings as the wide normal range of CSF cAMP values (14,15) . Therefore, we decided to examine neurologically intact rhesus monkeys in order to determine the presence or absence of intrinsic daily fluctuations in CSF cAlO? levels . Methods One female and two male rhesus monkeys (4 .5-6 .0 kg) were chronically implanted with Ommaya CSF reservoirs connected to silastic catheters placed in the fourth ventricle . This method permitted sterile aspirations of 0.5-1 .0 ml of CSF from the awake animal on a chronic basis . Details of this technique have been previously published (16) . Three mCi of 99mfc were injected into the reservoir followed by extensive, repeated manual compression and passive filling 445
44 6
Daily Variances in CSF cAMP Levels
Vol . 22, No . 6, 1978
of the reservoir . Radiocisternography of the ventricular system revealed CSF exchange between the ventricular system, the posterior fossa, and the upper spinal subarachnoid space . Computerized tomography revealed a neuroradiologically normal status with absence of hydrocephalus in all animals . To insure equilibration of CSF between the reservoir and the subarachnoid space, reservoirs were compressed and allowed to fill several times before sampling . Our initial studies demonstrated no significant loss in CSF cAMP content when CSF was allowed to incubate at 25 0C for 2 hours . This tends to discount possible decomposition of cAMP while in the reservoir despite reported occurrence of phosphodiesterase in CSF (17) . Animals were placed in chairs, enclosed in separate chambers, and fed regularly at 8 a .m . and 3 p .m . In the first experiences, monkeys were maintained on a 12-hour light/12-hour dark (12L/12D) lighting schedule (lighting was 20 foot-candles) . CSF samples (0 .5 ml) were taken every 3 hours over a 72-hour period . In later experiments, animals were adapted to continuous dim light (less than 0 .25 foot-candles) for 5 days before the 72-hour sampling period commenced . Aspirated samples were immediately frozen in liquid nitrogen They were later deproteinized with 0.5 ml of 6% trichloroand stored at -801C . acetic acid, ether-extracted to remove the acid, lyophilized, resuspended in buffer, and assayed radioimmunologically for cAMP content (18,19) . Radioimmunoassay kits were purchased from Schwartz-Mann Co ., Orangeburg, N . J . because intracranial CAMP may increase in stressed animals (20), the necessary restraint for and discomfort of withdrawing CSF from the Ommaya reservoirs of awake monkeys may have iatrogenically influenced the levels of cANP . Therefore, CSF samples were twice withdrawn from each of 4 monkeys both before and 1 hour after the induction of a light ketamine (10 mgm/kg) and atropine ( .004 mgm/kg) anesthesia . The preanesthesia value did not differ significantly from the 1 hour anesthetized value (Preanesthesia - 4 .9 _+ 1 .7 and anesthesia - 5 .9 _+ 2 .8 pmoles CAMP/ml CSF, respectively) . These results provide evidence that the observed fluctuations of CSF cAMP measured in this study were more likely due to factors other than a simple reflection of a stress induced to awake animals during withdrawal of CSF. Results and Discussion Part a of Figure 1 contains three records of monkey CSF cAMP content as a function of time during the 72-hour period, using animals exposed to a 12L/12D Fluctuations in cANP levels appear to be evident in-all light schedule . subjects . Considerable variability in cAMP levels in different subjects is suggested and is consistent with data on the wide range of cAMP levels in human CSF (14,15) . Part b of Figure 1 demonstrates that when the animals were maintained in continuous dim light for 5 days prior to the 72-hour dim light sampling period, a trend is evident toward a lower mean CAMP level in the dim light condition (18.9 pmoles cAMP/ml CSF in 12L/12D animals versus 10 .8 pmoles/ml in dim light animals) . This interpretation would be consistent with recent work on neural cyclic nucleotide rhythms influenced by lighting schedules (21-23) . In order to examine the variability of the cAMP measurements themselves, the coefficients of variation (S .D ./average) (24) of 9 pairs of calibration measurements from each of three different radioimmunoassay kits were averaged . The pooled coefficient was 0 .25 . One can obtain an estimate of the standard deviation at any cAMP concentration by multiplying 0 .25 by the concentration in pmoles of CAMP/ml CSF . For example, at 10 pmoles CAMP/ml CSF an estimate of the standard deviation (or measurement error) would be 2 .5 pmoles cAMP/ml CSF.
Daily Variances in OF cANP Levels
Vol . 22, No . 6, 1978
0600 90
1900
0600
TIME (hours) 0600 1800
1800
447
0600
90 70, 40
i
s 3: t
20
Y
10 0 70
0 l/24 D
46
ûE
n Y
20
10 0 0600
1900
0600
DAY 1 --
0600 1000 TIME (boon) DAY 2
1800
0600
DAY 3
FIG . 1 Cerebrospinal fluid cyclic AMP levels in rhesus Three monkeys were monkeys over a 72-hour period . accustomed either to a 12-hour light/12-hour dark schedule (12L/12D) as presented in (a) or to a 24hour dim light schedule (0 .25 foot-candles) (OL/24D) as shown in (b) . As stated above, the average cANP levels of dim light animals was 10 .8 (± 7 .3 S .D .), whereas for the light/dark animals the average was 18 .9 (+ 17 .2 S .D .) ; n - 71 for each mean . Since the S .D . of the light/dark animals (17 .2) is greater than that of the dim-light animals (7 .3), one would tend to believe that the observed fluctuations of CSF cAMP levels were not only significant with respect to measurement error, but also more prominent under light/dark conditions . Because of the nature of these experiments with primates, the lack of a large number of measurements does not allow one to perform a valid statistical test of these apparent differences .
44 8
Daily Variances in CSF cAMP Levels
Vol . 22, No . 6, 1978
When the results from all monkeys were evaluated no statistically significant dark/light differences for the 12-hour light/12-hour dark group were found . The Fried-n two-way analysis (25) was employed for this mathe matical examination. This statistical test is a non-parametric version of the analysis, of variance . It is insensitive to wild excursions of data, removes the caveat of correlated observations and adjusts for animal to animal differences. Possibly the lack of statistical significance might only mean that the sample size was too small to detect the differences suggested by the individual profiles in Figure 1 . In the future, the number of CSF samples might be increased by either using an automated, continuous 24-hour system of CSF withdrawal and collection, or the allocation of considerably greater numbers of enclosed, individual chambers for placement of additional numbers of animals. It is not clear from our experiments whether the changes we observed in CSF cAMP levels reflect changes in brain cANP, changes in transport of cMP from brain to CSF, degradation of CAMP within the intercellular spaces of the brain, or a combination of these factors . The potential influence of environmental lighting upon CSF cAMP concentration remains to be delineated . Knowledge of temporal fluctuations of CSF cANP levels demonstrated in our study may be useful in future experiments. Acknowledgment Special thanks are extended to Dr . Sheldon Levin and Mr . William Jackson of the Biostatistics Office of the Armed Forces Radiobiology Research Institute, Bethesda, Maryland, for their help in the statistical analysis of these data . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
J. B. SEBENS and J. KORF, Exp . Neurol . 46 333-344 (1975) . J. KORF, P. H . BOER, and D . FEKKES, Brain Res . 113 551-561 (1976) . J. W. KEBABIAN and P . GREENGARD, Science 174 1346-1349 (1971) . .SEGAL, and A. P . OLIVER, Adv. F. E. BLOOM, G. R . SIGGINS, B. J. HOFFER, M Cyclic Nucleotide Res . _5 603-618 (1975) . J. W . KEBABIAN, A. L . STEINER, and P . GREENGARD, J . Pharm. Exp. Ther . 19 3 474-488 (1975) . G. C. PALMER, F. SULSER, and G. A. ROBINSON, Neuropharmacology 12 327-337 (1973) . D. RUDMAN and J. W. ISAACS, Endocrinology 97 1476-1480 (1975) . V . V . MYLLA, E . R. HEIKKINEN, H . VAPAATALO, and E. HOKKANEN, Eur . Neurol . _13 123-130 (1975) . D . RUDMAN, A . FLEISCHER, and M. H. KITNER, New Engl . J . Med. 295 635-638 (1976) . D. RUDMAN, M. S. O'BRIEN, A. S . McKINLEY, J. C . HOFFMAN, and J . H.PATTERSON, J . Clin . Endocrinology Metab . 42 1088-1097 (1976) . K. M. A. WELCH, E . CHABI, J . H. NELL, K. BARTOSH, A. N . C . CHEE, N. T. MATTHEW, and V. S . ACHAR, Headache 16 160-167 (1976) . W. K. PHILLIP-DORMSTON, Pfluegers Arch . 362 223-227 (1976) . W. K. PHILLIP-DORMSTON and R . SIEGERT, Med . Microbiol . Immunol . (Berl .) 161 11-13 (1975) . B . B . BROOKS, J . SODÉ, W. K. ENGEL, Cyclic Nucleotide Metabolism in Neuromuscular Disease, presented at UCLA Conf . on Research Trends in Amyotrophic Lateral Sclerosis, Los Angeles, Calif., Oct 18-21 (1975) . V. V. MYLLA, V. VAPAATALO, E . HOKKANEN, and E . R. HEIKKINEN, Eur . Neurol . 12 28-32 (1972) . J . H. WOOD, D . G . POPLACK, W. A. BLEYER, and A . K. OMMAYA, Science 195 499501 (1977) .
Vol . 22, No . 6, 1978
Daily Variences in CSF cAMP Levels
449
17 . H . HIKAKA, M . SHIBUYA, T . ASANO, and F . HARA, J . Neurochem . 25 49-53 (1975) . 18 . A . L . STEINER, C . W . PARKER, and D . M . KIPNIS, J . Biol . Chem . 247 11061113 (1972) . 19 . A . L . STEINER, A . S . PAGLIARI, L . R . CHASE, and D . M .KKIPNIS, J . Biol . Chem. 274 1114-1120 (1972) . 20 .'i.L . DELAPAZ, S . R . DICKMAN, and B . I . GROSSER, Brain Research 85 171-175 (1975) . 21 . K . P . MINNEMAN and L . L . IVERSEN, Nature 260 59-61 (1976) . 22 . J . A . ROMERO, Fed . Proc . 35 1157-1161 (1976) . 23 . M . PERLO, B . FESTHOFF, M . EBERT, E . K . GORDON, M . G . ZIEGLER, C . R . LAKE, H . HOFFMAN, D . K . JOHNSON, and T . N . CHASE, Neuroscience Abstr . _2 499Soc . Neurosc ., Bethesda, Md . (1976) . 24 . R . G . D . STEE L and J . H . TORRIE, Principles and Procedures of Statistics , p . 20, McGraw-Hill (1960) . 25 . W . J . CONOVER, Practical Nonparametric Statistics p . 224, Wiley and Sons (1971) .
