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Normalization of dendritic spine numbers in rat hippocampus after termination of phenylacetate injections (PKU model)
354
Brain Research, 329 (1985) 354-355 Elsevier
BRE 20666
Normalization of dendritic spine numbers in rat hipp~ampus after termination of phenylacetate injections (PKU model) DANIEL J. LACEY Departments of Neurology and Pediatrics, State University of New York at Buffalo, Buffalo, NY 14222 (U.S.A.)
(Accepted October 23rd, 1984) Key words: pathologicseries of human phenylketonuria (PKU) - - rat model - - dendritic changes
Increased numbers of apical dendritic spines are present on hippocampal CA1 pyramidal cells in rats injected with phenylacetate from 2 to 21 days of life !f animals are sacrificed at 20-30 days. However, if sacrificed at 60-90 days, spine counts are not significantly different from saline injected controls. These results suggest that this increased spine density at 3-4 weeks represents retardation of normal maturational spine loss rather than an actual hyperplasia, and is reversible upon termination of the phenylacetate injections. Implications for human pathologicseries of phenylketonuria are discussed.
Pathologic series of human phenylketonuria (PKU) have until recently focused upon abnormalities of myelin and non-specific, relatively minor neuronal changes 1. Since 1980, 4 adults with PKU have been reported whose brains were studied by Golgi methods 1,a4. These have shown reductions in dendritic spine density on cortical pyramidal cells and truncated dendritic arborization patterns consistent with a maturational arrest. Animal models have been dedeveloped in the rat which closely simulate the biochemical, behavioral and pathologic features of human PKU t2. Although different methods have been used to induce hyperphenylalaninemia in rat pups, Golgi studies have shown smaller dendritic arbors and spine reduction in cortical pyramidal cells and cerebellar Purkinje cells 3,8,1°. We have previously reported an increased number of longer and thinner dendritic spines of CA1 hippocampal pyramidal cells in the stratum radiatum of hyperphenylalaninemic rats4, 5, using the Staten Island rat model of PKU described by Wen et a1.12. However, it was not clear whether the increased concentration of spines represented an actual hyperplasia or a failure of normal maturational spine reduction. We therefore injected rat pups with phenylacetate or saline, as above, from 2 to 21 days of life and sacrificed
different groups at 21, 30, 60 and 90 days of age. Brains were processed by Nissi and Golgi-Cox methods, sectioned at 10/¢m and 90/am, respectively. Spine counts per 50/~m were recorded for the primary and secondary apical dendrites of CA1 hippocampal pyramidal cells. For each age group, 10 well-impregnated pyramidal cells from each of 5 subjects were counted. Mean counts per 50/~m were compared by t-tests and analysis of variance. Spine morphology was qualitatively assessed. Results are presented in Table I. As previously reported, spine counts in the hippocapal stratum radiatum are significantly greater in animals sacrificed at 20-30 days. However, by 60 days and at 90 days of age, no significant numerical differences were evident. Qualitative observations of spine morphology and length revealed a reduction in the numbers of thin, long spines and replacement by spines with thicker heads and shorter stalks. Whether this represents experiential alteration in morphology, as described by Brandon and Coss in the honeybee, or a pre-programmed maturational evolution is uncertain z. Quantitative data regarding morphological alterations of spines with age in the postnatal rat are not available. Our results imply not only that delayed maturational spine reduction is present in this animal model of
Correspondence: D. J. Lacey, Division of Neurology,Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, NY 14222, U.S.A.
0006-8993/85/$03.30(~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
355 TABLE I
p h e n y l a l a n i n e m i a has two primary clinical implica-
Mean (standard deviation) numbers of hippocampal pyramidal cell dendritic spines per 50 l~m in stratum radiatum
tions for h u m a n PKU. Firstly, a teratogenic potential exists for fetuses born to mothers with uncontrolled P K U 7. At-risk infants, although not having classic P K U , have a high incidence of microcephaly, mental retardation and congenital anomalies. It appears that the likelihood of fetal d a m a g e relates directly to maternal phenylalanine levels early in gestation. W e have recently injected phenylacetate into pregnant rats and found abnormal cortical pyramidal cells in their offspring 6. Therefore, the p r o b l e m of ' m a t e r n a l P K U ' may be amenable to animal experimentation. Secondly, h u m a n studies have clearly shown that to escape the cognitive consequences of P K U , patients must be treated with a low phenylalanine diet in early infancy. W h e n , if ever, the diet can be discontinued is still not settled. W h e t h e r prevention of retardation by a diet in the child with P K U relates to normalization of dendritic spine counts in the rat h i p p o c a m p u s when h y p e r p h e n y l a l a n i n e m i a is t e r m i n a t e d is of course conjectural. H o w e v e r , maturational spine reduction does occur in human h i p p o c a m p u s and many h u m a n mental retardation syndromes have shown fetal-like dendritic spines present in affected patients9,10,13. Most of these human conditions are not currently treatable by enzyme r e p l a c e m e n t so that animal models are still necessary for experimental manipulation of relevant variables.
Location
Day of sacrifice 21
30
60
90
Primary apical dendrites CA1 52 (4.0) CA2 53 (4.2) b CA3 45
53 (4.4) 52 (4.2) b 44
46 44 40
43 42 35
Secondary apical dendrites CA1 51 (4.2) b CA2 48 (4.4) b CA3 47
52 (4.8) b 49 (4.2) b 46
49 46 46
47 42 40
Statistical significance compared to saline-injected controls: a p<0.05;b P<0.1.
P K U , but also that it is reversible. The h u m a n myelin and Golgi studies which have shown m a t u r a t i o n a l arrest in adult P K U patients are derived from persons who have generally had persistent hyperphenylalaninemia, i.e. u n t r e a t e d P K U 1. O u r animals received phenylacetate injections only until 21 days of age and none thereafter. W h e t h e r the increased spine density would persist if phenylacetate injections were continued until the time of sacrifice will be subsequently investigated. The timing of induction and termination of hyper1 Bauman, M. L. and Kemper, Th. L., Morphologic and histoanatomic observations of the brain in untreated human phenylketonuria, Acta neuropathol. (Berl.), 58 (1982) 55-63. 2 Brandon, J. G. and Coss, R. G., Rapid dendritic spine stem shortening during one-trial learning; the honeybee's first orientation flight, Brain Research, 252 (1982) 51. 3 Hogan, R. N., Dendritic alterations in brains of experimentally hyperphenylalaninemic rats, J. Neuropath. exp. NeuroL, 37 (1978) 628. 4 Lacey, D. J., Hippocampal dendritic abnormalities in a rat model of phenylketonuria, Neurology, 32 (1982) A171. 5 Lacey, D. J., Hippocampal dendritic abnormalities in a rat model of phenylketonuria (PKU), Ann. Neurol., 16 (1984) 577. 6 Lacey, D. J., Dendritic abnormalities of cortical pyramidal cells in rat pups born to hyperphenylalaninemic mothers: a new animal model of 'maternal phenylketonuria'. Ann. Neurol., 16 (1984) 384. 7 Lipson, A., Beuhler, B., Bart|ey, J., Walsh, D., Yu, J., O'Halloran, M. and Webster, W., Maternal hyperphenylalaninemia fetal effects, J. Pediatr., 104 (1984) 216-220. 8 Nigam, M. P. and Yagnik, P., The effect of hyperphenylalaninemia on rat neocortex. A Golgi study, Ann. Neurol., 6 (1979) !83.
9 Purpura, D. P., Normal and aberrant neuronal development in the cerebral cortex of human fetus and young infant. In N. A. Buchwald and M. A. B. Brazier (Eds.), Brain Mechanisms in Mental Retardation, Academic Press, New York, 1975, pp. 141-169. 10 Purpura, D. P., Pathobiology of cortical neurons in metabolic and unclassified amentias. In R. Katzman (Ed.), Congenital and Acquired Cognitive Disorders, Raven Press, New York, 1979, pp. 43-68. 11 Robain, O., Wen, G. Y., Wisniewski, H. M., Shek, J. W. and Loo, Y. H., Purkinje cell dendritic development in experimental phenylketonuria, Acta neuropathol. (Berl.), 53 (1981) 107-112. 12 Wen, G. Y., Wisniewski, H. M., Shek, J. W., Loo, Y. H. and Fulton, T. R., Neuropathology of phenylacetate poisoning in rats: an experimental model of phenylketonuria, Ann. Neurol., 7 (1980) 557-566. 13 Williams, R. S., Marshall, P. C. and Lott, I. T., Dendritic abnormalities in 'steely hair syndrome': a Golgi microscopic analysis, Neurology, 27 (1977) 369. 14 Williams, R. S., Hauser, S. L., Purpura, D. P., DeLong, G. R. and Swisher, C. N., Autism and mental retardation. Neuropathologic studies performed in four retarded persons with autistic features, Arch. Neurol., 37 (1980) 749-753.
Brain Research, 329 (1985) 354-355 Elsevier
BRE 20666
Normalization of dendritic spine numbers in rat hipp~ampus after termination of phenylacetate injections (PKU model) DANIEL J. LACEY Departments of Neurology and Pediatrics, State University of New York at Buffalo, Buffalo, NY 14222 (U.S.A.)
(Accepted October 23rd, 1984) Key words: pathologicseries of human phenylketonuria (PKU) - - rat model - - dendritic changes
Increased numbers of apical dendritic spines are present on hippocampal CA1 pyramidal cells in rats injected with phenylacetate from 2 to 21 days of life !f animals are sacrificed at 20-30 days. However, if sacrificed at 60-90 days, spine counts are not significantly different from saline injected controls. These results suggest that this increased spine density at 3-4 weeks represents retardation of normal maturational spine loss rather than an actual hyperplasia, and is reversible upon termination of the phenylacetate injections. Implications for human pathologicseries of phenylketonuria are discussed.
Pathologic series of human phenylketonuria (PKU) have until recently focused upon abnormalities of myelin and non-specific, relatively minor neuronal changes 1. Since 1980, 4 adults with PKU have been reported whose brains were studied by Golgi methods 1,a4. These have shown reductions in dendritic spine density on cortical pyramidal cells and truncated dendritic arborization patterns consistent with a maturational arrest. Animal models have been dedeveloped in the rat which closely simulate the biochemical, behavioral and pathologic features of human PKU t2. Although different methods have been used to induce hyperphenylalaninemia in rat pups, Golgi studies have shown smaller dendritic arbors and spine reduction in cortical pyramidal cells and cerebellar Purkinje cells 3,8,1°. We have previously reported an increased number of longer and thinner dendritic spines of CA1 hippocampal pyramidal cells in the stratum radiatum of hyperphenylalaninemic rats4, 5, using the Staten Island rat model of PKU described by Wen et a1.12. However, it was not clear whether the increased concentration of spines represented an actual hyperplasia or a failure of normal maturational spine reduction. We therefore injected rat pups with phenylacetate or saline, as above, from 2 to 21 days of life and sacrificed
different groups at 21, 30, 60 and 90 days of age. Brains were processed by Nissi and Golgi-Cox methods, sectioned at 10/¢m and 90/am, respectively. Spine counts per 50/~m were recorded for the primary and secondary apical dendrites of CA1 hippocampal pyramidal cells. For each age group, 10 well-impregnated pyramidal cells from each of 5 subjects were counted. Mean counts per 50/~m were compared by t-tests and analysis of variance. Spine morphology was qualitatively assessed. Results are presented in Table I. As previously reported, spine counts in the hippocapal stratum radiatum are significantly greater in animals sacrificed at 20-30 days. However, by 60 days and at 90 days of age, no significant numerical differences were evident. Qualitative observations of spine morphology and length revealed a reduction in the numbers of thin, long spines and replacement by spines with thicker heads and shorter stalks. Whether this represents experiential alteration in morphology, as described by Brandon and Coss in the honeybee, or a pre-programmed maturational evolution is uncertain z. Quantitative data regarding morphological alterations of spines with age in the postnatal rat are not available. Our results imply not only that delayed maturational spine reduction is present in this animal model of
Correspondence: D. J. Lacey, Division of Neurology,Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, NY 14222, U.S.A.
0006-8993/85/$03.30(~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
355 TABLE I
p h e n y l a l a n i n e m i a has two primary clinical implica-
Mean (standard deviation) numbers of hippocampal pyramidal cell dendritic spines per 50 l~m in stratum radiatum
tions for h u m a n PKU. Firstly, a teratogenic potential exists for fetuses born to mothers with uncontrolled P K U 7. At-risk infants, although not having classic P K U , have a high incidence of microcephaly, mental retardation and congenital anomalies. It appears that the likelihood of fetal d a m a g e relates directly to maternal phenylalanine levels early in gestation. W e have recently injected phenylacetate into pregnant rats and found abnormal cortical pyramidal cells in their offspring 6. Therefore, the p r o b l e m of ' m a t e r n a l P K U ' may be amenable to animal experimentation. Secondly, h u m a n studies have clearly shown that to escape the cognitive consequences of P K U , patients must be treated with a low phenylalanine diet in early infancy. W h e n , if ever, the diet can be discontinued is still not settled. W h e t h e r prevention of retardation by a diet in the child with P K U relates to normalization of dendritic spine counts in the rat h i p p o c a m p u s when h y p e r p h e n y l a l a n i n e m i a is t e r m i n a t e d is of course conjectural. H o w e v e r , maturational spine reduction does occur in human h i p p o c a m p u s and many h u m a n mental retardation syndromes have shown fetal-like dendritic spines present in affected patients9,10,13. Most of these human conditions are not currently treatable by enzyme r e p l a c e m e n t so that animal models are still necessary for experimental manipulation of relevant variables.
Location
Day of sacrifice 21
30
60
90
Primary apical dendrites CA1 52 (4.0) CA2 53 (4.2) b CA3 45
53 (4.4) 52 (4.2) b 44
46 44 40
43 42 35
Secondary apical dendrites CA1 51 (4.2) b CA2 48 (4.4) b CA3 47
52 (4.8) b 49 (4.2) b 46
49 46 46
47 42 40
Statistical significance compared to saline-injected controls: a p<0.05;b P<0.1.
P K U , but also that it is reversible. The h u m a n myelin and Golgi studies which have shown m a t u r a t i o n a l arrest in adult P K U patients are derived from persons who have generally had persistent hyperphenylalaninemia, i.e. u n t r e a t e d P K U 1. O u r animals received phenylacetate injections only until 21 days of age and none thereafter. W h e t h e r the increased spine density would persist if phenylacetate injections were continued until the time of sacrifice will be subsequently investigated. The timing of induction and termination of hyper1 Bauman, M. L. and Kemper, Th. L., Morphologic and histoanatomic observations of the brain in untreated human phenylketonuria, Acta neuropathol. (Berl.), 58 (1982) 55-63. 2 Brandon, J. G. and Coss, R. G., Rapid dendritic spine stem shortening during one-trial learning; the honeybee's first orientation flight, Brain Research, 252 (1982) 51. 3 Hogan, R. N., Dendritic alterations in brains of experimentally hyperphenylalaninemic rats, J. Neuropath. exp. NeuroL, 37 (1978) 628. 4 Lacey, D. J., Hippocampal dendritic abnormalities in a rat model of phenylketonuria, Neurology, 32 (1982) A171. 5 Lacey, D. J., Hippocampal dendritic abnormalities in a rat model of phenylketonuria (PKU), Ann. Neurol., 16 (1984) 577. 6 Lacey, D. J., Dendritic abnormalities of cortical pyramidal cells in rat pups born to hyperphenylalaninemic mothers: a new animal model of 'maternal phenylketonuria'. Ann. Neurol., 16 (1984) 384. 7 Lipson, A., Beuhler, B., Bart|ey, J., Walsh, D., Yu, J., O'Halloran, M. and Webster, W., Maternal hyperphenylalaninemia fetal effects, J. Pediatr., 104 (1984) 216-220. 8 Nigam, M. P. and Yagnik, P., The effect of hyperphenylalaninemia on rat neocortex. A Golgi study, Ann. Neurol., 6 (1979) !83.
9 Purpura, D. P., Normal and aberrant neuronal development in the cerebral cortex of human fetus and young infant. In N. A. Buchwald and M. A. B. Brazier (Eds.), Brain Mechanisms in Mental Retardation, Academic Press, New York, 1975, pp. 141-169. 10 Purpura, D. P., Pathobiology of cortical neurons in metabolic and unclassified amentias. In R. Katzman (Ed.), Congenital and Acquired Cognitive Disorders, Raven Press, New York, 1979, pp. 43-68. 11 Robain, O., Wen, G. Y., Wisniewski, H. M., Shek, J. W. and Loo, Y. H., Purkinje cell dendritic development in experimental phenylketonuria, Acta neuropathol. (Berl.), 53 (1981) 107-112. 12 Wen, G. Y., Wisniewski, H. M., Shek, J. W., Loo, Y. H. and Fulton, T. R., Neuropathology of phenylacetate poisoning in rats: an experimental model of phenylketonuria, Ann. Neurol., 7 (1980) 557-566. 13 Williams, R. S., Marshall, P. C. and Lott, I. T., Dendritic abnormalities in 'steely hair syndrome': a Golgi microscopic analysis, Neurology, 27 (1977) 369. 14 Williams, R. S., Hauser, S. L., Purpura, D. P., DeLong, G. R. and Swisher, C. N., Autism and mental retardation. Neuropathologic studies performed in four retarded persons with autistic features, Arch. Neurol., 37 (1980) 749-753.