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Histofluorescence evaluation of sympathetic nerve fibers in hearts of developing rats exposed to ethanol during gestation
Vol. 23, No. 9, pp. 1075-1079,1984 Printed in Great Britain
Neuropharmacology
0028-3908/84$3.00+ 0.00 Pergamon Press Ltd
HISTOFLUORESCENCE EVALUATION OF SYMPATHETIC NERVE FIBERS IN HEARTS OF DEVELOPING RATS EXPOSED TO ETHANOL DURING GESTATION PUSHPA V. THADANI* and M. R. WELLS Veterans Administration Medical Center and Department of Neurology, Georgetown University Medical School and Department of Physiology, George Washington University Medical School, Washington, D.C., U.S.A. (Accepted 1 December 1983) Summary-Histofluorescence for catecholamines pathetic innervation in the heart of control rats In all groups, sympathetic innervation began in lesser extent, in the myocardial surface between innervation progressed in density first in the atria
has been used to examine the ingrowth of the symand rats exposed either pre- or perinatally to ethanol. primary association with large blood vessels and, to a 2 and 6 days of postnatal age. From day 9 onward, and right ventricle. The deep muscle of the left ventricle
was least innervated of all areas of the heart at all time periods examined. In pups exposed continuously to ethanol or withdrawn at birth, the innervation pattern and density were similar to controls, although slight qualitative differencesmay have occurred in the first two weeks of the postnatal period in the group exposed continuously to ethanol. The reported altered cardiac biochemical responses to sympathetic agents after pre- or perinatal exposure to ethanol did not appear to be associated with significant alterations in the growth of sympathetic nerve fibers into the heart. It is suggested that the alterations in the development of the cardiac sympathetic nervous system induced by ethanol probably occurs at the level of nerve terminals or adrenergic receptors.
There is clinical evidence to indicate that children born of chronic alcoholic mothers show growth deficiency as well as abnormal EEG and unspecified cardiac defects (Havlicek and Childaeva, 1976; Jones, Smith, Streissguth and Myrianthopoulos, 1974; Jones and Smith, 1975; Streissguth, Landesman-Dwyer, Martin and Smith, 1980). In animals exposed pre- or perinatally to ethanol, the basal pattern of ornithine decarboxylase (ODC) in the heart as well as cardiac response induced by various adrenergic stimulants was altered during development (Bartolome, Schanberg and Slotkin, 1981; Thadani, Lau, Slotkin and Schanberg, 1977a; Thadani, Slotkin and Schanberg, 1977b; Thadani, 198313).These biochemical changes were associated with decreased weight of the heart. A recent study has shown that in rats, maternal consumption of ethanol does not produce profound changes in the uptake of [3H]norepinephrine into synaptic vesicles in the heart during development (Thadani, 1983a). To determine whether these changes in the development of the function of adrenergic nerves were due to the altered ingrowth of sympathetic nerve fibers in the heart, the present authors have examined catecholamine histofluorescence in the heart of rats whose mothers received ethanol during gestation and postpartum. Timed pregnant Sprague-Dawley rats (ZivicMiller Laboratories, Allison Park, Pennsylvania) *Present address: National Institute of Environmental Health Sciences, National Toxicology Program, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
were housed individually in breeding cages and on the 1Ith day of gestation were started on a nutritionally complete liquid diet (Sustacal@) without further access to water. On the 13th day of gestation, the animals were divided into three groups (matched by food intake and weight). One of the experimental groups received ethanol, 6% w/v, in Sustacal during gestation only, while the second group was given ethanol in Sustacal both during gestation and postpartum (continuous ethanol group). The remaining group was used as control and received Sustacal, made isocaloric and isonutritional to the ethanoldiet, by addition of sucrose. The group of mothers that received ethanol only during gestation were withdrawn from ethanol postpartum by the substitution of ethanol with sucrose in Sustacal (withdrawn at birth group). To ensure that all groups consumed the same amount, the control intake was restricted to that consumed by the groups given ethanol throughout the study. The intake of food was measured daily and averaged approx. 72 ml/day. The 13th day of gestation was chosen because earlier studies have shown that in rats, the autonomic nervous system in the heart starts to develop during this period and that the effect of ethanol on the developing heart is dependent upon the time of exposure during gestation (Robkin, Shepard and Dyer, 1976; Thadani et al., 1977b). At birth, pups in all groups were randomized and then redistributed to the respective nursing mothers. In every case, the size of litter was kept equivalent, 10-12 pups/mother. Maternal care appeared to be
1075
PUSHPAV. THADANI and
1076
normal in all groups. The pups were killed by decapitation at 2, 6, 9, 14 and 20 days of postnatal age (n = 2/group/each age) and the hearts were removed quickly and frozen immediately in liquid nitrogen. The frozen tissue was cut into 20 pm sections and the catecholamine examined sections were for fluorescence by the histochemical method of De LaTorre (1980). For the development of the catecholamine fluorescence, the sections (about 10) were placed on a cold slide and warmed just prior to dipping of the slide into SPG solution (0.2 M sucrose-0.236 M monobasic KH,PO,-1% glyoxylic acid monohydride, pH 7.4) at room temperature. The slides were dipped into SPG solution three times quickly and then dried immediately under a stream of cool air from a hair dryer for at least 20 min. After drying, 4-5 drops of light USP mineral oil were placed onto the sections and the slide with the sections was covered with a coverslip. The slides were warmed in a preheated oven at 95°C for 2.5 min. After placing fresh mineral oil and new coverslips, the sections were examined under a fluorescence microscope for the innervation of sympathetic nerve fibers into the developing heart. RESULTS AND DISCUSSlON
The development of catecholamine histofluorescence in the heart of control and rats exposed to ethanol throughout the postnatal period was similar, as depicted schematically in Fig. 1. The appearance of nerve fibers in 2-day-old rats was minimal and primarily associated with large blood vessels and, to a lesser extent, the myocardial surface.
0x
2 DAY
6
DAY
9 DAY
20 DAY
Fig. 1. Schematic diagram of the ingrowth of catecholaminergic fibers into the developing heart with increasing postnatal age. The innervation over the surface of the heart is shown on the left and a midventricular cross section is shown on the right. Relative changes in heart size are not demonstrated.
M. R. WELU
A significant increase in the nerve fibers primarily associated with the innervation of large blood vessels was observed between 6 and 9 days of postnatal age. At day 6, the innervation had occurred at the surface of the heart including the ventricular apex, the atria, and part of the nearby surface of the right ventricle. From postnatal day 9 onward, the catecholamine histofluorescence was seen in deeper regions of the atria and the right ventricle and it became denser with the increasing postnatal age, indicating that sympathetic innervation of the deeper myocardial layers of the atrium and right ventricle was occurring. The full innervation of the surface of the left ventricle did not occur until 14 days of postnatal age. At day 17 and 20, most of the increasing innervation was discernable in the myocardium of the left ventricle as deeper layers of muscle and the interventricular septum became progressively innervated. The lumen of the left ventricle had also begun to receive innervation during this postnatal period. In spite of the increase in the innervation of the myocardium of the left ventricule, some areas remained only sparsely innervated at 20 days of postnatal age. In pups exposed continuously to ethanol or withdrawn at birth, the development of catecholamine histofluorescence in the heart was similar. However, some minor qualitative differences were observed in the group given ethanol continuously and the controls or the withdrawn group. At 6 and 9 days, the density of nerve fibers in the plexus of fibers around blood vessels in the group given ethanol continuously appeared slightly greater than in the controls or the withdrawn group and at 14 days, slightly less (Fig. 2). These possible contrasts were not marked, however, and were near the limit of qualitative resolution for the method used. There was also some variability of innervation observed between animals in a group. The ingrowth of sympathetic nerve fibers observed in the present study for control animals corresponds with physiological studies demonstrating an increase of the heart rate response to sympathetic agents with postnatal age (Seidler and Slotkin, 1979; Standen, 1978). In pups exposed to ethanol during development, no significant differences in the growth of sympathetic nerve fibers could be detected in terms of timing or pattern which would clearly account for the observed differences in heart weight, cardiac ornithine decarboxylase activity and the cardiac biochemical responses to sympathetic stimulation in such animals (Bartolome et al., 1981; Thadani et al., 1977a, b;‘Thadani, 1983b). It should be noted, however, that even minor differences in the density of innervation in critical areas, such as the sinus and atrioventricular nodes, have the potential to produce considerable alteration in the cardiac response to sympathetic agents. The minor qualitative differences suggested in the current study could correspond to the ethanol-induced alterations. Thus, if alterations of the sympathetic innervation to the heart are responsible for the observed ethanol-induced changes,
Control
Continuous
Ethanol
6 Day
9 Day
14
Day
Fig. 2. Photomicrographs of contrasting innervation between control and animals exposed continuously to ethanol for the same areas of the right ventricle at 6, 9 and 14 days postnatal age. These examples represent some of the more extreme differences observed between groups. Innervation in other areas or between other animals in the groups was either similar or less striking. Photomicrographs of contrasting innervation for the withdrawn groups are not shown as the pattern of innervation was similar to that of the control group.
1077
Ethanol, development cardiac sympathetic nerves
the present data suggest that these changes are subtle, nf ~PTVP “1I IPI~PI ‘” ,_* “1 ..“‘ .V te-minalr i”.. 1.1..US”or adrenergic perhaps at the --fin I,...* p~3, ^^rL.... uuuu r-c,.. LUQU-* at +I., LUGF;~uJ:, ir;vtil of ingrowth recej-‘--,. or nerve fibers. In conclusion, these histofluorescence data demonstrate a vascular-related pattern of sympathetic innervation in the heart of the developing rat with the atria and right ventricle receiving innervation left ventricle.
While
no profound
before the
alterations
of in-
growth of svmoathetic nerve fibers could be detected between thkethanol-exposed and the control animals, subtle
differences
in the fine ramifications
of fibers
were suggested. These data indicate that the ethanolinduced pathetic
developmental axis probably
alterations
of the heart sym-
of growth
of nerve fibers.
occur at the level of the nerve terminals or adrenergic receptors rather than a gross
alteration
Acknowledgements-Supported by funds from a special emphasis program on Alcohol Research, Veterans Administration, Washington, D.C. The authors wish to thank Ms Nancy MitcheIl at NIEHS for typing this manusc~pt.
REFERENCES Bartolome J. V., Schanberg S. M. and Slotkin T. A. (1981) Premature development of cardiac sympathetic neurotransmission in the fetal alcohol syndrome. Life Sei. 28: 571-576.
De LaTorre J. C. (1980) An improved approach to histofluorescence using the SPG method for tissue monoamines. J. Neurosci. Meth. 3: l-5.
1079
Havlicek V. and Childaeva R. (1976) E.E.G. component of fetal alcohol syndrome. Lunc~r 2: 477. Jones K. L., Smith D. W., Streissguth A. P. and Myrianthopoulos M. C. (1974) Outcome in offspring of chronic alcoholic women. Lancet 1: 10761078. Jones K. L. and Smith D. W. (1975) The fetal alcohol syndrome. Teratology 12: I-10. Robkin M. A., Shepard T. H. and Dyer D. C. (1976) Autonomic receptors of the early rat embryo heart: Growth and development. Proc. Sot. exp. Bioi. Med. 151:
7994303. Seidler F. J. and Slotkin T. A. (1979) Presynaptic and postsynaptic contributions to ontogeny of sympathetic control of heart rate in the preweanling rat. Br. J. Pharmac. 65: 431434.
Standen N. B. (1978) The postnatal development of adrenoceptor response to agonists and electrical stimulation in rat isolated atria. Br. J. Pharmac. 64: 83-89. Streissguth A. P., Landesman-Dwyer S., Martin J. C. and Smith D. W. (1980) Teratogenic effects of alcohol in humans and laboratory animals. Science, Washington 209: 353-361.
Thadani P. V., Lau C., Slotkin T. A. and Schanberg S. M. (1977a) Effect of maternal ethanol ingestion on neonatal rat brain and heart ornithine decarboxylase. Biochem. Pharmac. 26: 523-527.
Thadani P. V., Slotkin T. A. and Schanberg S. M. (1977b) Effects of late or early postnatal ethanol exposure on ornithine decarboxyfase activity in brain and heart of develooine rats. ~euroDh~rmaco~o~v 16: 289-293. Thadani b. ?. (1983a) Ek;ect of mat&al ethanol administration on synaptic vesicles development in heart of rat offspring. ~e~ro~~~rrn~co~o~y 22z 931-933. Thadani P. V. (1983b)Prenatal ethanol alters development of cardiac ornithine decarboxylase response to adrenergic agents in rat. I. Continuous exposure. Archs int. Pharmacadyn. Ther. 265: 192-201.
Neuropharmacology
0028-3908/84$3.00+ 0.00 Pergamon Press Ltd
HISTOFLUORESCENCE EVALUATION OF SYMPATHETIC NERVE FIBERS IN HEARTS OF DEVELOPING RATS EXPOSED TO ETHANOL DURING GESTATION PUSHPA V. THADANI* and M. R. WELLS Veterans Administration Medical Center and Department of Neurology, Georgetown University Medical School and Department of Physiology, George Washington University Medical School, Washington, D.C., U.S.A. (Accepted 1 December 1983) Summary-Histofluorescence for catecholamines pathetic innervation in the heart of control rats In all groups, sympathetic innervation began in lesser extent, in the myocardial surface between innervation progressed in density first in the atria
has been used to examine the ingrowth of the symand rats exposed either pre- or perinatally to ethanol. primary association with large blood vessels and, to a 2 and 6 days of postnatal age. From day 9 onward, and right ventricle. The deep muscle of the left ventricle
was least innervated of all areas of the heart at all time periods examined. In pups exposed continuously to ethanol or withdrawn at birth, the innervation pattern and density were similar to controls, although slight qualitative differencesmay have occurred in the first two weeks of the postnatal period in the group exposed continuously to ethanol. The reported altered cardiac biochemical responses to sympathetic agents after pre- or perinatal exposure to ethanol did not appear to be associated with significant alterations in the growth of sympathetic nerve fibers into the heart. It is suggested that the alterations in the development of the cardiac sympathetic nervous system induced by ethanol probably occurs at the level of nerve terminals or adrenergic receptors.
There is clinical evidence to indicate that children born of chronic alcoholic mothers show growth deficiency as well as abnormal EEG and unspecified cardiac defects (Havlicek and Childaeva, 1976; Jones, Smith, Streissguth and Myrianthopoulos, 1974; Jones and Smith, 1975; Streissguth, Landesman-Dwyer, Martin and Smith, 1980). In animals exposed pre- or perinatally to ethanol, the basal pattern of ornithine decarboxylase (ODC) in the heart as well as cardiac response induced by various adrenergic stimulants was altered during development (Bartolome, Schanberg and Slotkin, 1981; Thadani, Lau, Slotkin and Schanberg, 1977a; Thadani, Slotkin and Schanberg, 1977b; Thadani, 198313).These biochemical changes were associated with decreased weight of the heart. A recent study has shown that in rats, maternal consumption of ethanol does not produce profound changes in the uptake of [3H]norepinephrine into synaptic vesicles in the heart during development (Thadani, 1983a). To determine whether these changes in the development of the function of adrenergic nerves were due to the altered ingrowth of sympathetic nerve fibers in the heart, the present authors have examined catecholamine histofluorescence in the heart of rats whose mothers received ethanol during gestation and postpartum. Timed pregnant Sprague-Dawley rats (ZivicMiller Laboratories, Allison Park, Pennsylvania) *Present address: National Institute of Environmental Health Sciences, National Toxicology Program, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
were housed individually in breeding cages and on the 1Ith day of gestation were started on a nutritionally complete liquid diet (Sustacal@) without further access to water. On the 13th day of gestation, the animals were divided into three groups (matched by food intake and weight). One of the experimental groups received ethanol, 6% w/v, in Sustacal during gestation only, while the second group was given ethanol in Sustacal both during gestation and postpartum (continuous ethanol group). The remaining group was used as control and received Sustacal, made isocaloric and isonutritional to the ethanoldiet, by addition of sucrose. The group of mothers that received ethanol only during gestation were withdrawn from ethanol postpartum by the substitution of ethanol with sucrose in Sustacal (withdrawn at birth group). To ensure that all groups consumed the same amount, the control intake was restricted to that consumed by the groups given ethanol throughout the study. The intake of food was measured daily and averaged approx. 72 ml/day. The 13th day of gestation was chosen because earlier studies have shown that in rats, the autonomic nervous system in the heart starts to develop during this period and that the effect of ethanol on the developing heart is dependent upon the time of exposure during gestation (Robkin, Shepard and Dyer, 1976; Thadani et al., 1977b). At birth, pups in all groups were randomized and then redistributed to the respective nursing mothers. In every case, the size of litter was kept equivalent, 10-12 pups/mother. Maternal care appeared to be
1075
PUSHPAV. THADANI and
1076
normal in all groups. The pups were killed by decapitation at 2, 6, 9, 14 and 20 days of postnatal age (n = 2/group/each age) and the hearts were removed quickly and frozen immediately in liquid nitrogen. The frozen tissue was cut into 20 pm sections and the catecholamine examined sections were for fluorescence by the histochemical method of De LaTorre (1980). For the development of the catecholamine fluorescence, the sections (about 10) were placed on a cold slide and warmed just prior to dipping of the slide into SPG solution (0.2 M sucrose-0.236 M monobasic KH,PO,-1% glyoxylic acid monohydride, pH 7.4) at room temperature. The slides were dipped into SPG solution three times quickly and then dried immediately under a stream of cool air from a hair dryer for at least 20 min. After drying, 4-5 drops of light USP mineral oil were placed onto the sections and the slide with the sections was covered with a coverslip. The slides were warmed in a preheated oven at 95°C for 2.5 min. After placing fresh mineral oil and new coverslips, the sections were examined under a fluorescence microscope for the innervation of sympathetic nerve fibers into the developing heart. RESULTS AND DISCUSSlON
The development of catecholamine histofluorescence in the heart of control and rats exposed to ethanol throughout the postnatal period was similar, as depicted schematically in Fig. 1. The appearance of nerve fibers in 2-day-old rats was minimal and primarily associated with large blood vessels and, to a lesser extent, the myocardial surface.
0x
2 DAY
6
DAY
9 DAY
20 DAY
Fig. 1. Schematic diagram of the ingrowth of catecholaminergic fibers into the developing heart with increasing postnatal age. The innervation over the surface of the heart is shown on the left and a midventricular cross section is shown on the right. Relative changes in heart size are not demonstrated.
M. R. WELU
A significant increase in the nerve fibers primarily associated with the innervation of large blood vessels was observed between 6 and 9 days of postnatal age. At day 6, the innervation had occurred at the surface of the heart including the ventricular apex, the atria, and part of the nearby surface of the right ventricle. From postnatal day 9 onward, the catecholamine histofluorescence was seen in deeper regions of the atria and the right ventricle and it became denser with the increasing postnatal age, indicating that sympathetic innervation of the deeper myocardial layers of the atrium and right ventricle was occurring. The full innervation of the surface of the left ventricle did not occur until 14 days of postnatal age. At day 17 and 20, most of the increasing innervation was discernable in the myocardium of the left ventricle as deeper layers of muscle and the interventricular septum became progressively innervated. The lumen of the left ventricle had also begun to receive innervation during this postnatal period. In spite of the increase in the innervation of the myocardium of the left ventricule, some areas remained only sparsely innervated at 20 days of postnatal age. In pups exposed continuously to ethanol or withdrawn at birth, the development of catecholamine histofluorescence in the heart was similar. However, some minor qualitative differences were observed in the group given ethanol continuously and the controls or the withdrawn group. At 6 and 9 days, the density of nerve fibers in the plexus of fibers around blood vessels in the group given ethanol continuously appeared slightly greater than in the controls or the withdrawn group and at 14 days, slightly less (Fig. 2). These possible contrasts were not marked, however, and were near the limit of qualitative resolution for the method used. There was also some variability of innervation observed between animals in a group. The ingrowth of sympathetic nerve fibers observed in the present study for control animals corresponds with physiological studies demonstrating an increase of the heart rate response to sympathetic agents with postnatal age (Seidler and Slotkin, 1979; Standen, 1978). In pups exposed to ethanol during development, no significant differences in the growth of sympathetic nerve fibers could be detected in terms of timing or pattern which would clearly account for the observed differences in heart weight, cardiac ornithine decarboxylase activity and the cardiac biochemical responses to sympathetic stimulation in such animals (Bartolome et al., 1981; Thadani et al., 1977a, b;‘Thadani, 1983b). It should be noted, however, that even minor differences in the density of innervation in critical areas, such as the sinus and atrioventricular nodes, have the potential to produce considerable alteration in the cardiac response to sympathetic agents. The minor qualitative differences suggested in the current study could correspond to the ethanol-induced alterations. Thus, if alterations of the sympathetic innervation to the heart are responsible for the observed ethanol-induced changes,
Control
Continuous
Ethanol
6 Day
9 Day
14
Day
Fig. 2. Photomicrographs of contrasting innervation between control and animals exposed continuously to ethanol for the same areas of the right ventricle at 6, 9 and 14 days postnatal age. These examples represent some of the more extreme differences observed between groups. Innervation in other areas or between other animals in the groups was either similar or less striking. Photomicrographs of contrasting innervation for the withdrawn groups are not shown as the pattern of innervation was similar to that of the control group.
1077
Ethanol, development cardiac sympathetic nerves
the present data suggest that these changes are subtle, nf ~PTVP “1I IPI~PI ‘” ,_* “1 ..“‘ .V te-minalr i”.. 1.1..US”or adrenergic perhaps at the --fin I,...* p~3, ^^rL.... uuuu r-c,.. LUQU-* at +I., LUGF;~uJ:, ir;vtil of ingrowth recej-‘--,. or nerve fibers. In conclusion, these histofluorescence data demonstrate a vascular-related pattern of sympathetic innervation in the heart of the developing rat with the atria and right ventricle receiving innervation left ventricle.
While
no profound
before the
alterations
of in-
growth of svmoathetic nerve fibers could be detected between thkethanol-exposed and the control animals, subtle
differences
in the fine ramifications
of fibers
were suggested. These data indicate that the ethanolinduced pathetic
developmental axis probably
alterations
of the heart sym-
of growth
of nerve fibers.
occur at the level of the nerve terminals or adrenergic receptors rather than a gross
alteration
Acknowledgements-Supported by funds from a special emphasis program on Alcohol Research, Veterans Administration, Washington, D.C. The authors wish to thank Ms Nancy MitcheIl at NIEHS for typing this manusc~pt.
REFERENCES Bartolome J. V., Schanberg S. M. and Slotkin T. A. (1981) Premature development of cardiac sympathetic neurotransmission in the fetal alcohol syndrome. Life Sei. 28: 571-576.
De LaTorre J. C. (1980) An improved approach to histofluorescence using the SPG method for tissue monoamines. J. Neurosci. Meth. 3: l-5.
1079
Havlicek V. and Childaeva R. (1976) E.E.G. component of fetal alcohol syndrome. Lunc~r 2: 477. Jones K. L., Smith D. W., Streissguth A. P. and Myrianthopoulos M. C. (1974) Outcome in offspring of chronic alcoholic women. Lancet 1: 10761078. Jones K. L. and Smith D. W. (1975) The fetal alcohol syndrome. Teratology 12: I-10. Robkin M. A., Shepard T. H. and Dyer D. C. (1976) Autonomic receptors of the early rat embryo heart: Growth and development. Proc. Sot. exp. Bioi. Med. 151:
7994303. Seidler F. J. and Slotkin T. A. (1979) Presynaptic and postsynaptic contributions to ontogeny of sympathetic control of heart rate in the preweanling rat. Br. J. Pharmac. 65: 431434.
Standen N. B. (1978) The postnatal development of adrenoceptor response to agonists and electrical stimulation in rat isolated atria. Br. J. Pharmac. 64: 83-89. Streissguth A. P., Landesman-Dwyer S., Martin J. C. and Smith D. W. (1980) Teratogenic effects of alcohol in humans and laboratory animals. Science, Washington 209: 353-361.
Thadani P. V., Lau C., Slotkin T. A. and Schanberg S. M. (1977a) Effect of maternal ethanol ingestion on neonatal rat brain and heart ornithine decarboxylase. Biochem. Pharmac. 26: 523-527.
Thadani P. V., Slotkin T. A. and Schanberg S. M. (1977b) Effects of late or early postnatal ethanol exposure on ornithine decarboxyfase activity in brain and heart of develooine rats. ~euroDh~rmaco~o~v 16: 289-293. Thadani b. ?. (1983a) Ek;ect of mat&al ethanol administration on synaptic vesicles development in heart of rat offspring. ~e~ro~~~rrn~co~o~y 22z 931-933. Thadani P. V. (1983b)Prenatal ethanol alters development of cardiac ornithine decarboxylase response to adrenergic agents in rat. I. Continuous exposure. Archs int. Pharmacadyn. Ther. 265: 192-201.