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Alteration in adenylate cyclase response to aminergic stimulation following neonatal x-irradiation
Brain Research Eullerin, Vol. 5, pp. 649-651. Printed in the U.S.A.
Alteration in Adenylate Cyclase Response to Aminergic Stimulation Following Neonatal X-Irradiation R. B. CHRONISTER,’
G. C. PALMER*
AND
L. GERBRANDTt
ofA~a~orny and Pharmacology,
~ep~rf~e~~s
Neurosciences
* U~~~~er~~~y of South Alabama, College of medicine Mobile, AL 36688 and Research Program, t Brookline, MA 02116 Received
CHRONISTER,
L. GERBRANDT. Alteration in ndenylate cvclase response aminergic RES. BULL. 64%651, 1980.-X-&radiation of the rat neonatal hippocampus produces severe alterations in the architectonic features of the mature hippocampus. The most prominent alterations is a marked depletion of the granule cells of the dentate gyrus, with a subsequent realignment of CA 4 cells. The present data also show that norepinephrine (NE), dopamine and histamine stimulation of adenylate cyclase activity is severely attenuated in the hippocampi of irradiated animals. This failure suggests that the NE fibers of irradiated subjects, al~ou~ normal in content of NE, are not functions in some of their NE-effector actions.
stimulation
x-irradiation
R.
10 June 1980
G. C. PALMER
following
Adenylate cyclase
Hippocampus
THE hippocampus is now recognized as an area of the brain which has a significant amount of postnatal development [l,Z]. Fu~he~ore, the mo~holo~cal development of the hippocampus is altered in several ways subsequent to neonatal x-irradiation. Besides the direct effects of x-irradiation in depleting the hippocampus of juxta-mitotic dentate granule cells and glial cells [3,4], the entorhinal, commissural and noradrenergic projections to the hippocampus appear to be redistributed as an indirect consequence of these i~adiation-induced depletions [5,7]. The present studies were undertaken to determine if these redistributed noradrenergic fibers, although normal in level of norepinephrine, are functionally normal in terms of coupling to neurohumorally-sensitive adenylate cyclase. METHOD
Neonatal rats (Long-Evans hooded) of both sexes were irradiated either bilaterally (N=6) or unilaterally (N=12) between the 2nd and 15th postnatal days as previously described [3,5]; briefly, on days 2, 3, rats received a 200-R dose, and on days 5, 7, 9, 11, 13, and 15, a 150-R dose of irradiation, each at 225 kV and at a rate of 40 R/min. An additional number (N=lO) were also sham-i~iated to serve as stress and handling controls. Furthermore, following unilateral irradiation, the non-irradiated side was used as a control. At maturity (greater than 200 g weight), several animals (both sham and irradiated) were fixed by perfusion with loo/o
‘Send reprint requests to: Robert B. Chronister,
Norepinephrine
Formalin or fixed by saline perfusion and immersion in 95% aicohol. The brains were embedded in celloidin, sectioned, and examined for architectonic alteration. The remai~ng rats were decapitated, the brains removed, and the whole dentate gyrus-CA fields (with some subiculum present) dissected into either cold glycylglycine buffer for adenylate cyclase assays or 0.25 M sucrose for cyclic AMP phosphodiesterase determinations. Previously described methodologies [S] were used for the assays, and all determinations were
done in duplicate. Adenylate cyclase activity was expressed as pmoles of cyclic AMP formedlmg proteinimin. Cyclic AMP phosphodiesterase activities were expressed as either pmoles (high Km, with or without the calcium-dependent regulator protein) or nmoles (low Km) of cyclic AMP hydrolyzed/mg proteinimin. RESULTS
Animals which received bilateral x-irradiation were smaller in body weight (275 * 15 g) than controls (465 + 10 g). Furthermore, the total area of the hippocampi of the bilateral animals were also noticeably smaller than those of the controls (Fig. 1). Note that, in this figure, the most obvious change is in the dentate gyrus. Also visible are increased scattering of neuronal perikarya of CA 1 and granule cells, and a rearrangement of hitar cells. In the cases of unilateral x-irradiation, the irradiated hippocampus was again smaller than on the non-irradiated hemisphere. In all cases, irradiation dramatically lowered adenylate
Ph.D., University of South Alabama, Department
Copyright @ 1980 ANKHO
of Anatomy, Mobile, AL 36688.
International Inc.-0361-9230/80/060649-03!400.80/0
cnRONISTER,
PALMER AND GERBRANDT
FIG. 1. Nissl stains of both x-irradiated (“A”) and control (“BY hippo-pi. The orientation and location of the tissue sections are comparable to each other (note medial habenuiar nucleus -h- on both). The brains were sectioned down the sagittal midline and placed together so that the final block was comprised of one hemisphere of an x-irradiated next to one hemisphere of a control. This minimizes processing variables. Also, note that “A” is at a higher magnification than “B. ” This is due to differences in brain sizes. Alcoholic fixation, celloidin embedding. Calibration mark-l mm. Abbreviations: D-dentate gyrus; h-nucleus habenularis medialis.
TABLE
1
CYCLIC AMP PHOSPHODIESTERASE
Conditions
IN RAT HIPPOCAMPUS
Enzyme activity Irradiated Controls
High Km*
17.5 + I.7
16.5 rt 2.6
High Km plus calcium-dependent regulator protein (3.42 pgLp)*
28.5 t 2.9
35.1 + 10.4
Low Kmt
58.8 f 4.7f
32.2 rt 6.3
*uMoles of cyclic AMP hydrolyz~/mg protei~min. ?nMoles of cyclic AMP hydrolyzedimg proteinimin. Wignificantly different than control @<0.025) N=4.
cyclase activation by three concentrations (W6, LO-“, 10-“M) of NE, dopamine and histamine (cf. Fig. 2). Due to the generally lower variability when the subjects were used as their own controls, this effect was most pronounced in the unilateral x-irradiated animals, but was still present to a significant degree (Student’s t test) at many hormone concen-
trations in the bilaterally x-irradiated hippocampi. Interestin&y, basal enzyme activity was not signi~cantIy different between any control or irradiated hippocampal tissue. In additional subjects, cyclic AMP phosphodiesterase was measured (Table 1) in both the control hippocampus and the side receiving irradiation. Neither the high Km nor the
ADENYLATE
n
CYCLASE
IRRADIATED
BILATERAL
651
AND X-IRRADIATION 0
CONTROL
IRRADIATED
HIPPOCAMPUS
high Km plus calcium-dependent regulator protein (calmodulin) forms of the enzyme appeared to be altered by the irradiation. However, under conditions of low substrate (low Km form), the irradiated hippocampus displayed a significantly increased ability to metabolize cyclic AMP. DISCUSSION
TTT
i#
2 % ;=i
UNILATERAL
T
IRRADIATED
HIPPOCAMPUS
LL
CONCENTRATION(MI NOREPINEPHRINE
DOPAMINE
HISTAMINE
FIG. 2. Effect of bilateral (top panel) or unilateral (bottom panel) irradiation on bippocampal adenylate cyclase responses to norepinephrine, dopamine and histamine. Percent stimulation of activity was calculated by subtracting basal activity from stimulated activity and dividing this figure by basal activity. The values are the mean f SEM percent stimulation of adenylate cyclase (315 ? 27 p moles of cyclic AMPimg proteitimin for all controls and 320 2 31 for all irradiated tissue). The number of individual experiments (each conducted in duplicate) are denotated at the bases of the respective
The present results document the direct effects of x-irradiation on granule cell depletion (i.e., note the near total loss of the infrapyramidal wing of the dentate gyrus in Fig. 1A as compared to Fig. 1B) which have been previously observed using these techniques in neonatal rats [3, 4, 5, 71. Furthermore, the observations of a reduction in adenylate cyclase and increase in phosphodiesterase activities extend similar results found after neonatal x-irradiation of the developing cerebellum [6]. However, in light of the normal levels of norepinephrine (NE) which have been observed within the hippocampus using other procedures and the limited distribution of NE fibers to granule cells [7], these results are expected. Such findings suggest that (1) total adenylate cyclase responsiveness in the hippocampus is more directly tied to the integrity of dentate granule and/or hilar cells than is apparent from the distributions of NE terminals, (2) the total number of available NE-receptor sites is greatly reduced even though the corresponding NE levels are normal, (3) the number of NE-receptor sites are normal, but NE sites are changed in conformation towards an inactive form, (4) NE-receptor sites are normal in density and conformation but fail to couple with nucleotide-regulatory (GTP-binding) and catalytic (adenylate cyclase) proteins, or (5) NE-receptor coupling with its regulatory and catalytic units is normal, but the adenylate cyclase enzyme, per se, is inactivated by irradiation. Further work, needed to determine the origins and developmental and anatomical specificity of these deficiencies on the induction of cyclic AMP by NE, is in progress.
ACKNOWLEDGEMENTS
Appreciation is expressed to S. Jo Palmer and S. Sharma for technical assistance, to the Epilepsy Foundation of America, the Scottish Rite Schizophrenia Foundation, and 1 R03 MH 32418 for research support.
control bar graphs.
REFERENCES Angevine, J. B., Jr. Time of neuron origin in the hippocampal region. autoradiographic study in the mouse. Expl Neural. 2: ^ . An _ _^ _-__ Suppl., l-70, lY65. A&man, J. and G. D. Das. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. camp. Neurol. 124: 31+336, 1%5. Bayer, S. A., R. L. Brunner, R. Hine and J. Altman. Behavioral effects of interference with the postnatal acquisition of hippocampal granule cells. Nature New Biol. 242: 222-224, 1973. 4. Bayer, S. A. and J. Altman. Hippocampal development in the rat: Cytogenesis and morphogenesis examined with autoradiography and low-level x-irradiation. J. camp. Neurol. 158: 5>80, 1974.
5. Gerbrandt, L. K., G. Rose, R. L. Wheeler and G. Lynch. Distribution of the perforant path following selective elimination of granule cells. Expl Neurol. 62: 122-132, 1978. 6. Hoffer, B. J., R. Freedman, D. J. Woodward, J. W. Daly and P. Skolnick. B-Adrenergic control of cyclic AMP-generating systems in cerebellum: pharmacological heterogeneity confirmed by destruction in interneurons. Expi Neural. 51: 653-667, 1976. 7. Moore, R. Y., B. Ziegler and S. A. Bayer. Monoamine neuron innervation of the hippocampal formation: alteration by neonatal irradiation. Expl Neural. 60: 318-326, 1978. 8. Palmer, G. C. Interactions of antiepileptic drugs on adenylate cyclase and phosphodiesterases in rat and mouse cerebrum. Expl Neural.
63: 322-355.
1979.
Alteration in Adenylate Cyclase Response to Aminergic Stimulation Following Neonatal X-Irradiation R. B. CHRONISTER,’
G. C. PALMER*
AND
L. GERBRANDTt
ofA~a~orny and Pharmacology,
~ep~rf~e~~s
Neurosciences
* U~~~~er~~~y of South Alabama, College of medicine Mobile, AL 36688 and Research Program, t Brookline, MA 02116 Received
CHRONISTER,
L. GERBRANDT. Alteration in ndenylate cvclase response aminergic RES. BULL. 64%651, 1980.-X-&radiation of the rat neonatal hippocampus produces severe alterations in the architectonic features of the mature hippocampus. The most prominent alterations is a marked depletion of the granule cells of the dentate gyrus, with a subsequent realignment of CA 4 cells. The present data also show that norepinephrine (NE), dopamine and histamine stimulation of adenylate cyclase activity is severely attenuated in the hippocampi of irradiated animals. This failure suggests that the NE fibers of irradiated subjects, al~ou~ normal in content of NE, are not functions in some of their NE-effector actions.
stimulation
x-irradiation
R.
10 June 1980
G. C. PALMER
following
Adenylate cyclase
Hippocampus
THE hippocampus is now recognized as an area of the brain which has a significant amount of postnatal development [l,Z]. Fu~he~ore, the mo~holo~cal development of the hippocampus is altered in several ways subsequent to neonatal x-irradiation. Besides the direct effects of x-irradiation in depleting the hippocampus of juxta-mitotic dentate granule cells and glial cells [3,4], the entorhinal, commissural and noradrenergic projections to the hippocampus appear to be redistributed as an indirect consequence of these i~adiation-induced depletions [5,7]. The present studies were undertaken to determine if these redistributed noradrenergic fibers, although normal in level of norepinephrine, are functionally normal in terms of coupling to neurohumorally-sensitive adenylate cyclase. METHOD
Neonatal rats (Long-Evans hooded) of both sexes were irradiated either bilaterally (N=6) or unilaterally (N=12) between the 2nd and 15th postnatal days as previously described [3,5]; briefly, on days 2, 3, rats received a 200-R dose, and on days 5, 7, 9, 11, 13, and 15, a 150-R dose of irradiation, each at 225 kV and at a rate of 40 R/min. An additional number (N=lO) were also sham-i~iated to serve as stress and handling controls. Furthermore, following unilateral irradiation, the non-irradiated side was used as a control. At maturity (greater than 200 g weight), several animals (both sham and irradiated) were fixed by perfusion with loo/o
‘Send reprint requests to: Robert B. Chronister,
Norepinephrine
Formalin or fixed by saline perfusion and immersion in 95% aicohol. The brains were embedded in celloidin, sectioned, and examined for architectonic alteration. The remai~ng rats were decapitated, the brains removed, and the whole dentate gyrus-CA fields (with some subiculum present) dissected into either cold glycylglycine buffer for adenylate cyclase assays or 0.25 M sucrose for cyclic AMP phosphodiesterase determinations. Previously described methodologies [S] were used for the assays, and all determinations were
done in duplicate. Adenylate cyclase activity was expressed as pmoles of cyclic AMP formedlmg proteinimin. Cyclic AMP phosphodiesterase activities were expressed as either pmoles (high Km, with or without the calcium-dependent regulator protein) or nmoles (low Km) of cyclic AMP hydrolyzed/mg proteinimin. RESULTS
Animals which received bilateral x-irradiation were smaller in body weight (275 * 15 g) than controls (465 + 10 g). Furthermore, the total area of the hippocampi of the bilateral animals were also noticeably smaller than those of the controls (Fig. 1). Note that, in this figure, the most obvious change is in the dentate gyrus. Also visible are increased scattering of neuronal perikarya of CA 1 and granule cells, and a rearrangement of hitar cells. In the cases of unilateral x-irradiation, the irradiated hippocampus was again smaller than on the non-irradiated hemisphere. In all cases, irradiation dramatically lowered adenylate
Ph.D., University of South Alabama, Department
Copyright @ 1980 ANKHO
of Anatomy, Mobile, AL 36688.
International Inc.-0361-9230/80/060649-03!400.80/0
cnRONISTER,
PALMER AND GERBRANDT
FIG. 1. Nissl stains of both x-irradiated (“A”) and control (“BY hippo-pi. The orientation and location of the tissue sections are comparable to each other (note medial habenuiar nucleus -h- on both). The brains were sectioned down the sagittal midline and placed together so that the final block was comprised of one hemisphere of an x-irradiated next to one hemisphere of a control. This minimizes processing variables. Also, note that “A” is at a higher magnification than “B. ” This is due to differences in brain sizes. Alcoholic fixation, celloidin embedding. Calibration mark-l mm. Abbreviations: D-dentate gyrus; h-nucleus habenularis medialis.
TABLE
1
CYCLIC AMP PHOSPHODIESTERASE
Conditions
IN RAT HIPPOCAMPUS
Enzyme activity Irradiated Controls
High Km*
17.5 + I.7
16.5 rt 2.6
High Km plus calcium-dependent regulator protein (3.42 pgLp)*
28.5 t 2.9
35.1 + 10.4
Low Kmt
58.8 f 4.7f
32.2 rt 6.3
*uMoles of cyclic AMP hydrolyz~/mg protei~min. ?nMoles of cyclic AMP hydrolyzedimg proteinimin. Wignificantly different than control @<0.025) N=4.
cyclase activation by three concentrations (W6, LO-“, 10-“M) of NE, dopamine and histamine (cf. Fig. 2). Due to the generally lower variability when the subjects were used as their own controls, this effect was most pronounced in the unilateral x-irradiated animals, but was still present to a significant degree (Student’s t test) at many hormone concen-
trations in the bilaterally x-irradiated hippocampi. Interestin&y, basal enzyme activity was not signi~cantIy different between any control or irradiated hippocampal tissue. In additional subjects, cyclic AMP phosphodiesterase was measured (Table 1) in both the control hippocampus and the side receiving irradiation. Neither the high Km nor the
ADENYLATE
n
CYCLASE
IRRADIATED
BILATERAL
651
AND X-IRRADIATION 0
CONTROL
IRRADIATED
HIPPOCAMPUS
high Km plus calcium-dependent regulator protein (calmodulin) forms of the enzyme appeared to be altered by the irradiation. However, under conditions of low substrate (low Km form), the irradiated hippocampus displayed a significantly increased ability to metabolize cyclic AMP. DISCUSSION
TTT
i#
2 % ;=i
UNILATERAL
T
IRRADIATED
HIPPOCAMPUS
LL
CONCENTRATION(MI NOREPINEPHRINE
DOPAMINE
HISTAMINE
FIG. 2. Effect of bilateral (top panel) or unilateral (bottom panel) irradiation on bippocampal adenylate cyclase responses to norepinephrine, dopamine and histamine. Percent stimulation of activity was calculated by subtracting basal activity from stimulated activity and dividing this figure by basal activity. The values are the mean f SEM percent stimulation of adenylate cyclase (315 ? 27 p moles of cyclic AMPimg proteitimin for all controls and 320 2 31 for all irradiated tissue). The number of individual experiments (each conducted in duplicate) are denotated at the bases of the respective
The present results document the direct effects of x-irradiation on granule cell depletion (i.e., note the near total loss of the infrapyramidal wing of the dentate gyrus in Fig. 1A as compared to Fig. 1B) which have been previously observed using these techniques in neonatal rats [3, 4, 5, 71. Furthermore, the observations of a reduction in adenylate cyclase and increase in phosphodiesterase activities extend similar results found after neonatal x-irradiation of the developing cerebellum [6]. However, in light of the normal levels of norepinephrine (NE) which have been observed within the hippocampus using other procedures and the limited distribution of NE fibers to granule cells [7], these results are expected. Such findings suggest that (1) total adenylate cyclase responsiveness in the hippocampus is more directly tied to the integrity of dentate granule and/or hilar cells than is apparent from the distributions of NE terminals, (2) the total number of available NE-receptor sites is greatly reduced even though the corresponding NE levels are normal, (3) the number of NE-receptor sites are normal, but NE sites are changed in conformation towards an inactive form, (4) NE-receptor sites are normal in density and conformation but fail to couple with nucleotide-regulatory (GTP-binding) and catalytic (adenylate cyclase) proteins, or (5) NE-receptor coupling with its regulatory and catalytic units is normal, but the adenylate cyclase enzyme, per se, is inactivated by irradiation. Further work, needed to determine the origins and developmental and anatomical specificity of these deficiencies on the induction of cyclic AMP by NE, is in progress.
ACKNOWLEDGEMENTS
Appreciation is expressed to S. Jo Palmer and S. Sharma for technical assistance, to the Epilepsy Foundation of America, the Scottish Rite Schizophrenia Foundation, and 1 R03 MH 32418 for research support.
control bar graphs.
REFERENCES Angevine, J. B., Jr. Time of neuron origin in the hippocampal region. autoradiographic study in the mouse. Expl Neural. 2: ^ . An _ _^ _-__ Suppl., l-70, lY65. A&man, J. and G. D. Das. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. camp. Neurol. 124: 31+336, 1%5. Bayer, S. A., R. L. Brunner, R. Hine and J. Altman. Behavioral effects of interference with the postnatal acquisition of hippocampal granule cells. Nature New Biol. 242: 222-224, 1973. 4. Bayer, S. A. and J. Altman. Hippocampal development in the rat: Cytogenesis and morphogenesis examined with autoradiography and low-level x-irradiation. J. camp. Neurol. 158: 5>80, 1974.
5. Gerbrandt, L. K., G. Rose, R. L. Wheeler and G. Lynch. Distribution of the perforant path following selective elimination of granule cells. Expl Neurol. 62: 122-132, 1978. 6. Hoffer, B. J., R. Freedman, D. J. Woodward, J. W. Daly and P. Skolnick. B-Adrenergic control of cyclic AMP-generating systems in cerebellum: pharmacological heterogeneity confirmed by destruction in interneurons. Expi Neural. 51: 653-667, 1976. 7. Moore, R. Y., B. Ziegler and S. A. Bayer. Monoamine neuron innervation of the hippocampal formation: alteration by neonatal irradiation. Expl Neural. 60: 318-326, 1978. 8. Palmer, G. C. Interactions of antiepileptic drugs on adenylate cyclase and phosphodiesterases in rat and mouse cerebrum. Expl Neural.
63: 322-355.
1979.