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Purkinje cell loss and the noradrenergic system in the cerebellum of pcd mutant mice
Rr&
Research BuNerin, Vol. 7, pp. 711-714,
1981. Printed in the
U.S.A.
Purkinje Cell Loss and the Noradrenergic System in the Cerebellum of pcd Mutant Mice BERNARDINO
GHETTI
Department of Pathology, Division of Neuropathology Indiana University School of Medicine, Indianapolis, IN 46223 AND
SUSAN
RAY W. FULLER, K. HEMRICK-LUECKE
The Lilly Research
Laboratories,
BARRY D. SAWYER, AND MICHAEL J. SCHMIDT
Eli Lilly and Company,
Received
18 March
Indianapolis,
IN 46285
1981
GHETTI, B., R. W. FULLER,
B. D. SAWYER, S. K. HEMRICK-LUECKE AND M. J. SCHMIDT. Purkinje cell loss ofpcd mutant mice. BRAIN RES. BULL. 7(6) 711-714, 1981.-I’urkinje cells in the cerebellum receive inhibitory noradrenergic input from the locus coeruleus. In pcd mutant mice all F’urkinje cells degenerate by 45 days of age. The purpose of the present studies was to determine if the loss of these cerebellar neurons affects the amounts of norepinephrine in the cerebellum of mice 25-280 days of age. No significant changes in norepinephrine content were detected during or after F’urkinje cell degeneration. However, since degeneration led to a reduction in cerebellar weight, the norepinephrine concentration was increased in pcd mutants. These results indicate that despite the loss of a major postsynaptic target (F’urkinje cells), the cerebellar noradrenergic input remains stable.
and the noradrenergic
Purkinje cell
system in the cerebellum
pcd Mutant
Cerebellum
Degeneration
NOREPINEPHRINE in the cerebellum is contained in axons derived from noradrenergic neurons of the locus coeruleus and other pontine areas [2,111. Noradrenergic terminals in the cerebellar cortex form synaptic contacts with Purkinje cell dendrites and dendritic spines [l], and there is evidence that stimulation of the locus coeruleus inhibits Purkinje cell firing through an elevation of cyclic AMP in Purkinje cells [18]. There are few studies of the response of the noradrenergic system to alterations in cerebellar circuitry. A number of strains of mutant mice develop abnormalities in cerebellar structure [17], and in several strains (weaver, reeler, staggerer) the noradrenergic system has been studied with histochemical and biochemical methods [9]. The weaver mutation is characterized by a failure of the granule cells to migrate and their degeneration during the first postnatal week [ 121; the reeler by an aberrant position of Purkinje cells and granule cells relative to each other [16]; and the staggerer by an alteration of the Purkinje cells’ tertiary spines, the failure of Purkinje cells to establish contacts with granule cells, and granule cell degeneration [19,20]. Landis et al. [9] reported that the concentration of norepinephrine was elevated in the cerebellum of reeler and staggerer mutants but unchanged in the weaver cerebellum. However, only in the weaver and staggerer mice was the absolute amount of norepinephrine altered. Furthermore they
Copyright
0 1981 ANKHO
International
Norepinephrine
suggested that the aberrant noradrenergic terminal morphology seen in the cerebellar cortex of staggerer was linked somehow to Purkinje cell abnormalities. Although in the above mentioned strains of mice there are various degrees of Purkinje cell abnormalities, in no case do Purkinje cells degenerate [16]. Therefore, the fate of the noradrenergic system following complete loss of Purkinje cells is not known. We undertook experiments to determine if complete loss and absence of Purkinje cells would affect the noradrenergic input to the cerebellum at various ages of maturity [14]. We also attempted to determine the specificity of such changes by measuring norepinephrine levels in the cerebrum and brain stem. We used Purkinje cell degeneration (pcd) mice for these studies since, following normal development, complete loss of Purkinje cells occurs between 18 and 45 days of age [lo]. Ataxia appears between 22 and 24 days of age. Loss of Purkinje cells and later loss of granule cells are responsible for a progressive atrophy of the cerebellum [6]. METHOD
A colony of mice was established at Indiana University Medical Center from a stock of 10 couples of mice of the C57BL/6J strain, heterozygous for the gene pcd, originating from the Jackson Laboratory (Bar Harbor, ME). Mice homozygous for the Purkinje cell degeneration mutation
Inc.-0361-9230/81/12071
l-04$00.90/0
GHETTI El
712
TABLE CEREBELLAR
1
NOREPINEPHRINE IN CONTROL PCD MUTANT MICE
AND
pMoles Norepinephrine per Cerebellum Age (days) 2.5 31 74 92 180 280
Control 106.5 i 133.7 f 131.2 2 108.4 + 92.5 + 107.3 r
5.8 8.7 9.2 3.8 5.1 2.3
pcd 99.7 148.2 145.5 150.9 113.5 126.7
+ 6.1 t 12.4 2 13.5 ‘-’ 8.41 -+ 6.0* i- 17.7
Values represent the mean t SEM of 5 controls or 5 pcd mice at each age. Data are expressed in terms of norepinephrine per cerebellum. Values significantly different than “control” are indicated: *<0.05, t
were obtained either by intercrossing heterozygous pairs or by intercrossing heterozygous males with homozygous (pcd/pcd) mutant females. Heterozygotes (+/pcd) are free of neurologic symptoms and do not lose Purkinje cells. They were used as controls. At the different ages specified (see Table l), mice were decapitated and brains were quickly excised and dissected. Cerebellum was removed first, then the cerebrum was separated,from the brain stem with a cut through the superior colliculus. The tissue samples were frozen on dry ice and stored at - 15°C. Analyses were performed within a few days after the mice were killed. However, due to the difficulty in obtaining sizeable numbers of mice of each age on a given date,it was not possible to include all ages simultaneously for longitudinal studies. All mice within a single age group were killed on the same day, and the chemical analysis or cerebellum, brain stem and cerebrum was performed at the same time. Norepinephrine was determined by liquid chromatography with electrochemical detection [S]. RESULTS
The total amount of norepinephrine in the cerebellum of pcd mice was slightly higher than in control mice at all ages except 25 days of age, but differences were significant only at 92 (+39 percent) and 180 (+23 percent) days of age (Table 1). The data are expressed as pmoles norepinephrine per cerebellum rather than per unit weight or per mg protein because of the above-mentioned degeneration of nonnorepinephrine-neuronal elements in the cerebellum in the pcd mice. During the time span studied (25-280 days of age) the weight of the cerebellum declined progressively in the affected animals, while cerebellar weight was constant in control mice (Fig. 1). The greatest difference in weight was observed in the oldest mice where weights of 23 mg and 55 mg were found in affected and control mice, respectively. Since cerebellar weight declined in the pcd mice while norepinephrine levels increased slightly, the net effect was a marked and significant increase in norepinephrine concentrutions in pcd mice compared to control animals (Fig. 1). This increase was progressive, beginning with a + 17 percent
Al..
difference at 25 days of age and reaching a maximum of a + 180 percent difference in mice 280 days old. Similar changes, although not as dramatic, were observed in the brain stem of pcd mice (Table 2). Absolute amounts of norepinephrine were higher in the pcd than age-matched controls at all time points, and concentrations were also markedly elevated in the brain stem of pcd mutants. No consistent differences in brain stem weight were observed between control and pcd mice (data not shown), although at 280 days of age the brain stem of pcd mutants weighed 22 percent less than age matched controls. Absolute levels of norepinephrine in the cerebrum were similar in control and affected animals (Table 3). In this brain region a small (- 14 percent to -26 percent) but significant difference in weight was seen, leading to significantly higher amine concentrations in the mutant mice. DISCUSSION
Two major anatomical events characterize the pcd mutant: (I) the loss of F’urkinje cells which begins at 17 days and is complete by 45-50 days of age [lo], and (II) a severe loss of granule cells which is evident after 180 days of age and is advanced by 280 days of age [6]. The purpose of the present studies was to determine if the loss of these cerebellar neurons affected the amounts of norepinephrine in the cerebellum. In our studies few significant changes in norepinephrine content (norepinephrine per cerebellum) were detected during Purkinje cell degeneration or long after the disappearance of these target cells of the noradrenergic axons. However, since the sequence of degenerative events led to a marked reduction in cerebellar weight, the norepinephrine concentration increased in pcd mutants. The stability of the noradrenergic system in the pcd cerebellum contrasts with the marked reduction of other neurotransmitters that are released presynaptically to Purkinje cells. Following F’urkinje cell degeneration there is a significant fall in glutamate and aspartate (Ghetti and McBride, in preparation). These findings correlate more closely with the loss of parallel fibers and climbing fibers in the molecular layer of pcd mice. The present experiments do not reveal why norepinephrine remains unchanged in the cerebellum of pcd mice, or where cerebellar norepinephrine is localized. Norepinephrine might be reduced in the cerebellar cortex, but, due to sprouting of axon collaterals in the deep cerebellar nuclei, amounts of norepinephrine could be increased in the subcortical regions. Thus, toto1 norepinephrine in the whole cerebellum would be unchanged. However, when the cerebellar cortex was separated from deep nuclei, cortical norepinephrine was not decreased but was elevated [ 131. Furthermore, preliminary histofluorescence studies with the glyoxylic method (Ghetti et al. unpublished observations) indicated that the fluorescent varicosities in the cerebellar cortex of 120-day-old mutants are more dense than in controls. At this time it is not known whether norepinephrine in the cerebellar cortex of pcd mutants performs any function since Purkinje cells, the targets of the noradrenergic axons, are gone. It is conceivable that noradrenergic axons are also presynaptic to other cortical nerve cells; or norepinephrine axons could establish new contacts with neurons that normally do not receive noradrenergic input. Alternatively, norepinephrine axons that normally are presynaptic to Purkinje cells, might become apposed to the ghal elements which engulfed Purkinje cell debris during degeneration and influence their function. Noradrenergic @receptors have been detected on
NORADRENERGIC
713
SYSTEM IN pcd MUTANT
CONTROL
I :
50
100
150
100
280 50 ME IdiiysJ
150
280
FIG. 1. Cerebellar weight (solid line) and norepinephrine concentration (dashed line) in control and pcd mutant mice. Values represent the mean k SEM of 5 animals at each age.
TABLE 3
TABLE 2 BRAINSTEM
NOREPINEPHRINE IN CONTROL PCD MUTANT MICE
II Percent Change Norepinephrine Concentration
I pMoles Norepinephrine per Brainstem Age
(days) 25 31 74 92 180 280
Control 217.0 240.9 233.0 263.3 186.9 220.0
” 7.8 + 15.6 + 8.8 f 20.9 t 7.0 + 9.6
pcd 283.9 262.6 319.4 302.7 356.3 276.0
+ + ‘t f L
13.0* 18.4 14.8 26.0 15.8* 17.6
CEREBRAL
AND
+198 +44* +26* +30* +46t +62t
NOREPINEPHRINE IN CONTROL PCD MUTANT MICE
I pMoles Norepinephrine per Cerebrum Age (days) 25 31 74 92 180 280
Control 131.9 152.1 181.5 185.4 216.4 244.1
AND
II Percent Change Norepinephrine Concentration
pcd
zk 9.35 k 5.27 2 6.71 t 5.98 k 12.25 2 6.68
123.6 168.3 192.6 197.7 229.5 258.6
k 1.67 k 9.69 2 3.71 2 5.49 f 8.51 k 15.13
+7 +28* +18t +24* +30* +44*
Values represent the mean 2 SEM of 5 controls or 5 pcd mice at each age. Data are expressed in terms of norepinephrine per brainstem (column I) and percent change in norepinephrine concentration (column II). Values significantly different than “control” are indicated: *<0.05, tc0.01, ~
Values represent the mean f SEM of 5 controls or 5 pcd mice at each age. Data are expressed in terms of norepinephrine per cerebrum (column I) and percent change in norepinephrine concentration (column II). Values significantly different than “control” are indicated: *
glioma cells [7] and there is evidence that fi-receptors are also present in normal glial cells [8,22]. Furthermore, catecholamines caused a marked stimulation of cyclic AMP synthesis in cultures of glial cells [3, 7, 81. Elevation of cyclic AMP in glioma cell cultures 143,or exposure of glial cells to a biologically active derivative of cyclic AMP [211 stimulates process formation. Also, /3-adrenergic receptor stimulation of glioma cells increases the synthesis and release of nerve growth factor-like material [lS]. Thus, norepinephrine de-
rived from presynaptic terminals might provide a message glial cells to become activated and initiate the processes
phagocytosis
following neuronal
to
of
injury.
ACKNOWLEDGEMENTS
We appreciate the expert technical assistance provided by Ms. Constance Alyea and Mr. Donovan Pearson. This study was supported in part by Grant No. PHS ROl NS 14426 to B. Ghetti.
714
GHETTI
E7’ AL.
REFERENCES
1. Bloom, F. E., B. J. Hoffer and G. R. Siggins. Studies on norepinephrine containing afferents to cerebellar Purkinje cells of rat cerebellum. Bruin Res. 25: 501-521, 1971. 2. Chu, N. S. and F. E. Bloom. The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections. Brrrin Res. 66: l-21, 1974. 3. Clark, R. B., R. Gross, Y. F. Su and J. P. Perkins. Regulation of
adenosine 3’:5’-monophosphate content in human astrocytoma cells by adenosine and the adenine nucleotides. J. biol. Chem. 249: 52965303, 1974. 4. Edstrom, A., M. Kanje,
P. Lofgren and E. Walum. Druginduced alterations in morphology and level of CAMP in cultured human glioma cells. Exp. Cell Res. 95: 359-364, 1975. 5. Fuller, R. W. and K. W. Perry. Lowering of epinephrine concentration in rat brain by 2,3-dichloro-a-methylbenzylamine, an inhibitor of norepinephrine N-methyltransferase. B&hem. Pharmuc.
26: 2087-2090,
1977.
6. Ghetti, B., C. J. Alyea and J. Muller. Studies on the Purkinje
cell degeneration (pcd) mutant: primary pathology and transneuronal changes. J. Neuroprrth. exp. Neural. 37: 617, 1978. 7. Gilman, A. G. and M. Nirenberg. Effect of catecholamines on the adenosine 3’:S’cyclic monophosphate concentration of clonal satellite cells of neurons. Proc. ncrtn. Accrd. Sci. U.S.A. 68: 2165-2168, 1971. 8. Gilman, A. G. and B. K. Schrier. Adenosine cyclic 3’,5’monophosphate in fetal rat brain cell cultures. I. Effect of catecholamines. Mole. Phnrmctc. 8: 410-416, 1972. 9. Landis, S. C., W. J. Shoemaker, M. Schlumpf and F. E. Bloom. Catecholamines in mutant mouse cerebellum: fluorescence microscopic and chemical studies. Bruin Res. 93: 253266, 1975. 10. Mullen, R. J., E. M. Either and R. L. Sidman. Purkinje cell degeneration, a new neurological mutation in the mouse. Proc. nutn. Acud. SC.;. U.S.A. 73: 208-212. 1976. 11. Olson, L. and K. Fuxe. On the projection from the locus coeruleus noradrenaline neurons: the cerebellar innervation. Bruin Rr.\. 28: 1655171, 1971.
12. Rakic, P. and R. L. Sidman. Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice. J. camp. Neural. 152: 103-132, 1973. 13. Roffler-Tarlov, S. and M. J. Zigmond. Effects of Purkinje cell degeneration on the noradrenergic projection to mouse cerebellar cortex. Sot. Neurosci. Abst. 6: 600, 1980. 14. Sawyer, B. D., S. Hemrick-Luecke, M. J. Schmidt, R. W. Fuller and B. Ghetti. Catecholamine and cyclic nucleotide concentrations in the cerebellum of pcd mutant mice. Sot. Neurosci. Ahst. 6: 442, 1980. 1.5. Schwartz, J. P. and E. Costa. Regulation of nerve growth factor content in C-6 glioma cells by P-adrenergic receptor stimulation. Naunyn-Schmiedeberg’s Arch. Pharmac. 300: 123-129, 1977. 16. Sidman, R. L. Development of intemeuronal connections in brains of mutant mice. In: Physiological and Biochemical Aspects oy Nervous Integration, edited by F. C. Carlson.
Englewood Cliffs, New Jersey: Prentice-Hall, Inc.. 1968. pp. 163-193. 17. Sidman, R. L., M. D. Green and S. H. Appel. Ctrtalog of the Neurological Mutants of the Mouse. Cambridge, Massachusetts: Harvard Univ. Press, 1965. 18. S&ins, G. R., E. F. Battenberg, B. J. Hoffer, F. E. Bloom and A. L. Steiner. Noradrenergic stimulation of cyclic adenosine monophosphate in rat Purkinje neurons: an immunocytochemical study. Science 179: 585-588, 1973. 19. Sotelo. C. Dendritic abnormalities of Purkinie cells in the cerebellum of neurologic mutant mice (Weaver and Staggerer). In: Advances in Neurology, Vol. 12, edited by G. W. Kreutzberg. New York: Raven Press, 1975, pp. 335-351. 20. Sotelo, C. and J. P. Changeux. Transsynaptic degeneration “en cascade” in the cerebellar cortex of staggerer mutant mice. Brain Res. 67: 519-526, 1974. 21. Steinbach, J. H. and D. Schubert.
Multiple modes of dibutyryl cyclic AMP-induced process formation by clonal nerve and glial cells. Exp. Cell Res. 91: 44%453, 1975. 22. Van Calker, D. and B. Hamprecht. Effects of neurohormones on glial cells. In: Advunces in Cellulur Neurobiology. Vol. 1, edited by S. Fedoroff and L. Hertz. New York: Academic Press. 1980, pp. 31-67.
Research BuNerin, Vol. 7, pp. 711-714,
1981. Printed in the
U.S.A.
Purkinje Cell Loss and the Noradrenergic System in the Cerebellum of pcd Mutant Mice BERNARDINO
GHETTI
Department of Pathology, Division of Neuropathology Indiana University School of Medicine, Indianapolis, IN 46223 AND
SUSAN
RAY W. FULLER, K. HEMRICK-LUECKE
The Lilly Research
Laboratories,
BARRY D. SAWYER, AND MICHAEL J. SCHMIDT
Eli Lilly and Company,
Received
18 March
Indianapolis,
IN 46285
1981
GHETTI, B., R. W. FULLER,
B. D. SAWYER, S. K. HEMRICK-LUECKE AND M. J. SCHMIDT. Purkinje cell loss ofpcd mutant mice. BRAIN RES. BULL. 7(6) 711-714, 1981.-I’urkinje cells in the cerebellum receive inhibitory noradrenergic input from the locus coeruleus. In pcd mutant mice all F’urkinje cells degenerate by 45 days of age. The purpose of the present studies was to determine if the loss of these cerebellar neurons affects the amounts of norepinephrine in the cerebellum of mice 25-280 days of age. No significant changes in norepinephrine content were detected during or after F’urkinje cell degeneration. However, since degeneration led to a reduction in cerebellar weight, the norepinephrine concentration was increased in pcd mutants. These results indicate that despite the loss of a major postsynaptic target (F’urkinje cells), the cerebellar noradrenergic input remains stable.
and the noradrenergic
Purkinje cell
system in the cerebellum
pcd Mutant
Cerebellum
Degeneration
NOREPINEPHRINE in the cerebellum is contained in axons derived from noradrenergic neurons of the locus coeruleus and other pontine areas [2,111. Noradrenergic terminals in the cerebellar cortex form synaptic contacts with Purkinje cell dendrites and dendritic spines [l], and there is evidence that stimulation of the locus coeruleus inhibits Purkinje cell firing through an elevation of cyclic AMP in Purkinje cells [18]. There are few studies of the response of the noradrenergic system to alterations in cerebellar circuitry. A number of strains of mutant mice develop abnormalities in cerebellar structure [17], and in several strains (weaver, reeler, staggerer) the noradrenergic system has been studied with histochemical and biochemical methods [9]. The weaver mutation is characterized by a failure of the granule cells to migrate and their degeneration during the first postnatal week [ 121; the reeler by an aberrant position of Purkinje cells and granule cells relative to each other [16]; and the staggerer by an alteration of the Purkinje cells’ tertiary spines, the failure of Purkinje cells to establish contacts with granule cells, and granule cell degeneration [19,20]. Landis et al. [9] reported that the concentration of norepinephrine was elevated in the cerebellum of reeler and staggerer mutants but unchanged in the weaver cerebellum. However, only in the weaver and staggerer mice was the absolute amount of norepinephrine altered. Furthermore they
Copyright
0 1981 ANKHO
International
Norepinephrine
suggested that the aberrant noradrenergic terminal morphology seen in the cerebellar cortex of staggerer was linked somehow to Purkinje cell abnormalities. Although in the above mentioned strains of mice there are various degrees of Purkinje cell abnormalities, in no case do Purkinje cells degenerate [16]. Therefore, the fate of the noradrenergic system following complete loss of Purkinje cells is not known. We undertook experiments to determine if complete loss and absence of Purkinje cells would affect the noradrenergic input to the cerebellum at various ages of maturity [14]. We also attempted to determine the specificity of such changes by measuring norepinephrine levels in the cerebrum and brain stem. We used Purkinje cell degeneration (pcd) mice for these studies since, following normal development, complete loss of Purkinje cells occurs between 18 and 45 days of age [lo]. Ataxia appears between 22 and 24 days of age. Loss of Purkinje cells and later loss of granule cells are responsible for a progressive atrophy of the cerebellum [6]. METHOD
A colony of mice was established at Indiana University Medical Center from a stock of 10 couples of mice of the C57BL/6J strain, heterozygous for the gene pcd, originating from the Jackson Laboratory (Bar Harbor, ME). Mice homozygous for the Purkinje cell degeneration mutation
Inc.-0361-9230/81/12071
l-04$00.90/0
GHETTI El
712
TABLE CEREBELLAR
1
NOREPINEPHRINE IN CONTROL PCD MUTANT MICE
AND
pMoles Norepinephrine per Cerebellum Age (days) 2.5 31 74 92 180 280
Control 106.5 i 133.7 f 131.2 2 108.4 + 92.5 + 107.3 r
5.8 8.7 9.2 3.8 5.1 2.3
pcd 99.7 148.2 145.5 150.9 113.5 126.7
+ 6.1 t 12.4 2 13.5 ‘-’ 8.41 -+ 6.0* i- 17.7
Values represent the mean t SEM of 5 controls or 5 pcd mice at each age. Data are expressed in terms of norepinephrine per cerebellum. Values significantly different than “control” are indicated: *<0.05, t
were obtained either by intercrossing heterozygous pairs or by intercrossing heterozygous males with homozygous (pcd/pcd) mutant females. Heterozygotes (+/pcd) are free of neurologic symptoms and do not lose Purkinje cells. They were used as controls. At the different ages specified (see Table l), mice were decapitated and brains were quickly excised and dissected. Cerebellum was removed first, then the cerebrum was separated,from the brain stem with a cut through the superior colliculus. The tissue samples were frozen on dry ice and stored at - 15°C. Analyses were performed within a few days after the mice were killed. However, due to the difficulty in obtaining sizeable numbers of mice of each age on a given date,it was not possible to include all ages simultaneously for longitudinal studies. All mice within a single age group were killed on the same day, and the chemical analysis or cerebellum, brain stem and cerebrum was performed at the same time. Norepinephrine was determined by liquid chromatography with electrochemical detection [S]. RESULTS
The total amount of norepinephrine in the cerebellum of pcd mice was slightly higher than in control mice at all ages except 25 days of age, but differences were significant only at 92 (+39 percent) and 180 (+23 percent) days of age (Table 1). The data are expressed as pmoles norepinephrine per cerebellum rather than per unit weight or per mg protein because of the above-mentioned degeneration of nonnorepinephrine-neuronal elements in the cerebellum in the pcd mice. During the time span studied (25-280 days of age) the weight of the cerebellum declined progressively in the affected animals, while cerebellar weight was constant in control mice (Fig. 1). The greatest difference in weight was observed in the oldest mice where weights of 23 mg and 55 mg were found in affected and control mice, respectively. Since cerebellar weight declined in the pcd mice while norepinephrine levels increased slightly, the net effect was a marked and significant increase in norepinephrine concentrutions in pcd mice compared to control animals (Fig. 1). This increase was progressive, beginning with a + 17 percent
Al..
difference at 25 days of age and reaching a maximum of a + 180 percent difference in mice 280 days old. Similar changes, although not as dramatic, were observed in the brain stem of pcd mice (Table 2). Absolute amounts of norepinephrine were higher in the pcd than age-matched controls at all time points, and concentrations were also markedly elevated in the brain stem of pcd mutants. No consistent differences in brain stem weight were observed between control and pcd mice (data not shown), although at 280 days of age the brain stem of pcd mutants weighed 22 percent less than age matched controls. Absolute levels of norepinephrine in the cerebrum were similar in control and affected animals (Table 3). In this brain region a small (- 14 percent to -26 percent) but significant difference in weight was seen, leading to significantly higher amine concentrations in the mutant mice. DISCUSSION
Two major anatomical events characterize the pcd mutant: (I) the loss of F’urkinje cells which begins at 17 days and is complete by 45-50 days of age [lo], and (II) a severe loss of granule cells which is evident after 180 days of age and is advanced by 280 days of age [6]. The purpose of the present studies was to determine if the loss of these cerebellar neurons affected the amounts of norepinephrine in the cerebellum. In our studies few significant changes in norepinephrine content (norepinephrine per cerebellum) were detected during Purkinje cell degeneration or long after the disappearance of these target cells of the noradrenergic axons. However, since the sequence of degenerative events led to a marked reduction in cerebellar weight, the norepinephrine concentration increased in pcd mutants. The stability of the noradrenergic system in the pcd cerebellum contrasts with the marked reduction of other neurotransmitters that are released presynaptically to Purkinje cells. Following F’urkinje cell degeneration there is a significant fall in glutamate and aspartate (Ghetti and McBride, in preparation). These findings correlate more closely with the loss of parallel fibers and climbing fibers in the molecular layer of pcd mice. The present experiments do not reveal why norepinephrine remains unchanged in the cerebellum of pcd mice, or where cerebellar norepinephrine is localized. Norepinephrine might be reduced in the cerebellar cortex, but, due to sprouting of axon collaterals in the deep cerebellar nuclei, amounts of norepinephrine could be increased in the subcortical regions. Thus, toto1 norepinephrine in the whole cerebellum would be unchanged. However, when the cerebellar cortex was separated from deep nuclei, cortical norepinephrine was not decreased but was elevated [ 131. Furthermore, preliminary histofluorescence studies with the glyoxylic method (Ghetti et al. unpublished observations) indicated that the fluorescent varicosities in the cerebellar cortex of 120-day-old mutants are more dense than in controls. At this time it is not known whether norepinephrine in the cerebellar cortex of pcd mutants performs any function since Purkinje cells, the targets of the noradrenergic axons, are gone. It is conceivable that noradrenergic axons are also presynaptic to other cortical nerve cells; or norepinephrine axons could establish new contacts with neurons that normally do not receive noradrenergic input. Alternatively, norepinephrine axons that normally are presynaptic to Purkinje cells, might become apposed to the ghal elements which engulfed Purkinje cell debris during degeneration and influence their function. Noradrenergic @receptors have been detected on
NORADRENERGIC
713
SYSTEM IN pcd MUTANT
CONTROL
I :
50
100
150
100
280 50 ME IdiiysJ
150
280
FIG. 1. Cerebellar weight (solid line) and norepinephrine concentration (dashed line) in control and pcd mutant mice. Values represent the mean k SEM of 5 animals at each age.
TABLE 3
TABLE 2 BRAINSTEM
NOREPINEPHRINE IN CONTROL PCD MUTANT MICE
II Percent Change Norepinephrine Concentration
I pMoles Norepinephrine per Brainstem Age
(days) 25 31 74 92 180 280
Control 217.0 240.9 233.0 263.3 186.9 220.0
” 7.8 + 15.6 + 8.8 f 20.9 t 7.0 + 9.6
pcd 283.9 262.6 319.4 302.7 356.3 276.0
+ + ‘t f L
13.0* 18.4 14.8 26.0 15.8* 17.6
CEREBRAL
AND
+198 +44* +26* +30* +46t +62t
NOREPINEPHRINE IN CONTROL PCD MUTANT MICE
I pMoles Norepinephrine per Cerebrum Age (days) 25 31 74 92 180 280
Control 131.9 152.1 181.5 185.4 216.4 244.1
AND
II Percent Change Norepinephrine Concentration
pcd
zk 9.35 k 5.27 2 6.71 t 5.98 k 12.25 2 6.68
123.6 168.3 192.6 197.7 229.5 258.6
k 1.67 k 9.69 2 3.71 2 5.49 f 8.51 k 15.13
+7 +28* +18t +24* +30* +44*
Values represent the mean 2 SEM of 5 controls or 5 pcd mice at each age. Data are expressed in terms of norepinephrine per brainstem (column I) and percent change in norepinephrine concentration (column II). Values significantly different than “control” are indicated: *<0.05, tc0.01, ~
Values represent the mean f SEM of 5 controls or 5 pcd mice at each age. Data are expressed in terms of norepinephrine per cerebrum (column I) and percent change in norepinephrine concentration (column II). Values significantly different than “control” are indicated: *
glioma cells [7] and there is evidence that fi-receptors are also present in normal glial cells [8,22]. Furthermore, catecholamines caused a marked stimulation of cyclic AMP synthesis in cultures of glial cells [3, 7, 81. Elevation of cyclic AMP in glioma cell cultures 143,or exposure of glial cells to a biologically active derivative of cyclic AMP [211 stimulates process formation. Also, /3-adrenergic receptor stimulation of glioma cells increases the synthesis and release of nerve growth factor-like material [lS]. Thus, norepinephrine de-
rived from presynaptic terminals might provide a message glial cells to become activated and initiate the processes
phagocytosis
following neuronal
to
of
injury.
ACKNOWLEDGEMENTS
We appreciate the expert technical assistance provided by Ms. Constance Alyea and Mr. Donovan Pearson. This study was supported in part by Grant No. PHS ROl NS 14426 to B. Ghetti.
714
GHETTI
E7’ AL.
REFERENCES
1. Bloom, F. E., B. J. Hoffer and G. R. Siggins. Studies on norepinephrine containing afferents to cerebellar Purkinje cells of rat cerebellum. Bruin Res. 25: 501-521, 1971. 2. Chu, N. S. and F. E. Bloom. The catecholamine-containing neurons in the cat dorsolateral pontine tegmentum: distribution of the cell bodies and some axonal projections. Brrrin Res. 66: l-21, 1974. 3. Clark, R. B., R. Gross, Y. F. Su and J. P. Perkins. Regulation of
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