Immunohistochemical localization of N-acetylaspartate in rat brain.

N-acetylaspartate (NAA) is one of the most prevalent compounds in the mammalian nervous system. As such, NAA largely contributes to the major peak on water-suppressed proton magnetic resonance spectra. Highly specific antibodies to NAA demonstrate that this compound is discretely localized in a substantial number of neurons throughout the extent of the rat CNS. N-acetylaspartylglutamate (NAAG) is a structurally related neuronal dipeptide which is less widely distributed than NAA. NAAG and NAA immunoreactivities were extensively colocalized in many brainstem areas, where NAAG containing neurons were more numerous than in forebrain structures.     

Immunohistochemical localization of citrullinated proteins in adult rat brain

By using hybridoma technology, an IgM monoclonal antibody (F95) against multiple citrullinated synthetic and natural peptides was recently developed and used to stain immunohistochemically subsets of astrocytes and myelin basic protein (MBP) from selected regions of human brain (Nicholas and Whitaker [2002] Glia 37:328-336). With this antibody, the present study provides a more detailed localization of citrullinated epitopes in the central nervous system (CNS) by examining immunohistochemical staining patterns for F95 in the normal adult rat brain. Thus, immunohistochemical labeling for citrullinated epitopes was seen in white matter areas consistent with myelin staining; however, in general, it was more prominent and uniform in the caudal CNS (spinal cord, medulla oblongata, pons, and cerebellum) than in more rostral areas. F95 staining was also seen in cells and fibers often intimately associated with blood vessels and/or ventricular surfaces. By using dual-color immunofluorescence, the vast majority of this latter staining was colocalized within a subset of astrocytes also immunoreactive for glial fibrillary acidic protein (GFAP). By using Western blot analysis of rat brain proteins, multiple GFAP- and MBP-immunoreactive proteins and peptide fragments were seen, and many of them were also reactive with the F95 antibody. Thus, the present study not only demonstrates that citrullinated epitopes in normal rat brain are most concentrated in subsets of myelin and astrocytes but also provides evidence that GFAP, like MBP, may be present as multiple citrullinated isoforms.     

Immunohistochemical localization of superoxide dismutase in congenital hydrocephalic rat brain

The effects of active oxygen species in the development of congenital hydrocephalus have been investigated. Superoxide dismutase (SOD) is one of the scavengers of active oxygen species and there have been many recent reports on the relationship between neurological disorders by active oxygen species following reperfusion for ischemic brain and SOD. In this study, the localization of Cu-SOD and Zn-SOD in WIC-Hyd congenitally hydrocephalic rat brains was identified by the enzyme unlabeled antibody method. We examined the localization of SOD in the choroid plexus, hippocampus, and ependymal cells of the lateral ventricle and aqueduct of WIC-Hyd rats. SOD was hardly observed in the choroid plexus and faintly localized in the hippocampus and ependymal cells of the congenitally hydrocephalic brain, but was observed equally in the cytoplasm of the choroid plexus, hippocampus, and ependymal cells in control animals. In the hippocampus, less SOD was found in hydrocephalic rats than in controls. The SOD was slightly observed in the CA1 pyramidal cells in hydrocephalic rats. In the lateral ventricle and aqueductal ependyma, less SOD was found in hydrocephalic than in control rats. The amount of Cu, Zn-SOD in the congenitally hydrocephalic rat brain was less than in the control, especially in the choroid plexus. Therefore, we suspect that the production of SOD is congenitally reduced in the congenitally hydrocephalic rat brain, and this may promote the impairment of the function of choroid plexus and cilia due to increased active oxygen species. The reduction of SOD in the choroid plexus, hippocampus and ependymal cells of ventricles or aqueduct may promote the development of hydrocephalus in the congenitally hydrocephalic rat.     

Immunohistochemical localization of hyaluronic acid in rat and human brain

The immunohistochemical localization of hyaluronic acid (HA) was studied in rat and human brain using the monoclonal antibody NDOG1, which specifically recognizes HA. In both rat and human brain, HA-like immunoreactivity formed characteristic coats around neurons in highly selective areas. The staining was abolished by pretreatment of sections with testicular andStreptomyces hyaluronidases, indicating that the staining was specific for HA. In rat brain, positive neurons were located in the cerebral cortex, subiculum, amygdala, thalamic reticular nucleus, nuclei of the inferior colliculus, nuclei of the trapezoid body, and vestibular nuclei. They were also scattered in the hypothalamus, substantia nigra pars reticularis, red nucleus, parabrachial nuclei, brainstem reticular nuclear group, ventral cochlear nucleus, nuclei of lateral lemniscus, and deep cerebellar nuclei. Double immunohistochemical studies showed that many neurons staining for HA were positive for parvalbumin, with minor exceptions in the amygdala and piriform cortex, where some HA-positive neurons were also positive for calbindin-D28k. In the areas studied in human brain, the distribution of HA-positive neurons was virtually identical to that in rat brain. HA-positive neurons were not significantly altered in Alzheimer disease (AD) brain, suggesting that these neurons are resistant to the pathological process of AD.     

Immunohistochemical localization of the neural cannabinoid receptor in rat brain

The cannabinoid receptor family consists of two inhibitory G-protein-coupled receptors, CB1 and CB2. CB1 is distributed primarily in neural tissue, whereas CB2 is distributed predominately in immune cells. The distribution of cannabinoid receptors in neural tissue has been demonstrated by using ligand binding autoradiography with CP55,940, a high-affinity cannabinoid receptor ligand, and in situ hybridization. However, the localization of CB1 within individual cells in the brain remains to be defined. In the present study, domain-specific polyclonal antibody to amino acids 83–98 of CB1 was used to define the expression of the neural cannabinoid receptor at the histochemical level. The use of CB1-specific antiserum is advantageous in view of recent reports that CB2 also is expressed in the brain and binds CP55,940. Thus, utilization of anti-CB1 antiserum would allow for the specific detection of CB1 protein expression. The regional staining pattern for CB1 in rat brain was consistent with that reported for CB1 using ligand binding autoradiography and in situ hybridization. Intense immunoreactivity was present in the hippocampal formation, the basal ganglia, and the molecular layer of the cerebellum. Moderate immunohistochemical staining was observed in the olfactory bulb, piriform cortex, cerebral cortex, and the granular layer of the cerebellum. In addition, immunoreactive staining was concentrated on afferent projections and dendritic processes of neuronal cells and was present within cell bodies and on cell surfaces. These data indicate that the anti-CB1 antibody is a sensitive probe for the unequivocal histological discrimination of CB1 protein expression. J. Neurosci. Res. 51:391–402, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical localization of PDE10A in the rat brain

PDE10A is a newly identified cAMP/cGMP phosphodiesterase for which mRNA is highly expressed in the mammalian striatum. In the present study, PDE10A protein and mRNA expression throughout the rat brain were determined, using a monoclonal antibody (24F3.F11) for Western blot and immunohistochemical analyses and an antisense riboprobe for in situ hybridization. High levels of mRNA are observed in most of the neuronal cell bodies of striatal complex (caudate n, n. accumbens and olfactory tubercle), indicating that PDE10A is expressed by the striatal medium spiny neurons. PDE10A-like immunoreactivity is dense throughout the striatal neuropil, as well as in the internal capsule, globus pallidus, and substantia nigra. These latter regions lack significant expression of PDE10A mRNA. Thus, PDE10A is transported throughout the dendritic tree and down the axons to the terminals of the medium spiny neurons. These data suggest a role for PDE10A in regulating activity within both the striatonigral and striatopallidal pathways. In addition, PDE10A immunoreactivity and mRNA are found at lower levels in the hippocampal pyramidal cell layer, dentate granule cell layer and throughout the cortex and cerebellar granule cell layer. Immunoreactivity is detected only in cell bodies in these latter regions. This more restricted subcellular localization of PDE10A outside the striatum suggests a second, distinct function for the enzyme in these regions.     

Immunohistochemical localization of caffeine-induced c-Fos protein expression in the rat brain

Although caffeine is the most widely used central nervous system stimulant, the neuronal populations and pathways mediating its stimulant effects are not well understood. Using c-Fos protein as a marker for neuronal activation, the present study investigated the pattern of c-Fos induction at 2 hours after low locomotor-stimulant doses (1, 5, 10, and 30 mg/kg, i.p.) of caffeine and compared them with those after a higher dose (75 mg/kg, i.p.) or saline injection in adult male rats. Fos-immunoreactive neurons were counted in selected nuclei across the entire brain. Caffeine induced an increase in locomotor activity in a dose-dependent manner up to doses of 30 mg/kg and a decline at 75 mg/kg. Quantitative analysis of Fos-immunoreactive neurons indicated that no structures showed significant Fos expression at doses below 75mg/kg or a biphasic pattern of Fos expression, as in locomotion. In contrast, caffeine at 75 mg/kg induced a significant increase compared with the saline condition in the number of Fos-immunoreactive neurons in the majority of structures examined. The structures included the striatum, nucleus accumbens, globus pallidus, and substantia nigra pars reticulata and autonomic and limbic structures including the basolateral and central nuclei of the amygdala, paraventricular and supraoptic hypothalamic nuclei, periventricular hypothalamus, paraventricular thalamic nuclei, parabrachial nuclei, locus coeruleus, and nucleus of the solitary tract. The locomotor-enhancing effects of low doses of caffeine did not appear to be associated with significant Fos expression in the rat brain. J. Comp. Neurol. 401:89–108, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical localization of enkephalin in rat brain and spinal cord.

The distribution of immunoreactive enkephalin in rat brain and spinal cord was studied by immunoperoxidase staining using antiserum to leucine-enkephalin ([Leu5]-enkephalin) or methionine-enkephalin ([Met5]-enkephalin). Immunoreactive staining for both enkephalins was similarly observed in nerve fibers, terminals and cell bodies in many regions of the central nervous system. Staining of perikarya was detected in hypophysectomized rats or colchicine pretreated rats. The regions of localization for enkephalin fibers and terminals include in the forebrain: lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions of the cortex, corpus striatum, bed nucleus of the stria terminalis, hypothalamus including median eminence, thalamus and subthalamus; in the midbrain: nucleus interpeduncularis, periaqueductal gray and reticular formation; in the hind brain: nucleus parabrachialis, locus ceruleus, nuclei raphes, nucleus cochlearis, nucleus tractus solitarii, nucleus spinalis nervi trigemini, motor nuclei of certain cranial nerves, nucleus commissuralis and formatio reticularis; and in the spinal cord the substantia gelatinosa. In contrast enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephalon, mesencephalon and rhombencephalon. The results of the histochemical staining show that certain structures which positively stain for enkephalin closely correspond to the distribution of opiate receptors in the brain and thus support the concept that the endogenous opiate peptides are involved in the perception of pain and analgesia. The localization of enkephalin in the preoptic-hypothalamic region together with the presence of enkephalin perikarya in the paraventricular and supraoptic nuclei suggest a role of enkephalin in the regulation of neuroendocrine functions.     

Free radical-induced elevation of ornithine decarboxylase activity in developing rat brain slices

Objective. In developing brain, we have previously shown both in vivo [L.D. Longo, S. Packianathan, J.A. McQueary, R.B. Stagg, C.V. Byus and C.D. Cain, Acute hypoxia increases ornithine decarboxylase activity and polyamine concentrations in fetal rat brain, Proc. Natl. Acad. Sci. USA, Vol. 90 (1993) 692–696] and in vitro [S. Packianathan, C.D. Cain, B.H. Liwnicz and L.D. Longo, Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices, Brain Res., Vol. 688 (1995) 61–71] that acute hypoxia is associated with a significant increase in ornithine decarboxylase (ODC) activity and polyamine concentrations. We tested the hypothesis that oxygen free radicals induce an increase in ODC activity similar to that of hypoxia and that both this and the hypoxia-induced response are inhibited by free radical scavengers. Materials and methods. Slices of cerebrum, 300–500 μm thick, were made from P3 newborn Sprague-Dawley rat pups and equilibrated for 1 h in artificial cerebrospinal fluid continuously bubbled with 95% O₂/5% CO₂. Free radical-induced ODC activity response was measured beginning after a 1-h recovery period. Experiments were performed on slices treated with 5×10⁻⁷ M xanthine (X)+10 mU/ml xanthine oxidase (XO), with or without the free radical scavengers superoxide dismutase (SOD; 100 U/ml), catalase (CAT; 700 U/ml) or glutathione peroxidase (GPX; 3 U/ml). We also quantified slice malonaldehyde concentrations in response to hypoxia (21% O₂/5% CO₂/74% N₂). Results. Under control conditions, ODC activity was stable during the 2-h post-recovery period. In response to X/XO treatment, ODC activity increased 2.3-fold at 1.5 h post-recovery. In examining ODC activity as a function of xanthine dose, we noted that ODC activity increased in response to 2.5×10⁻⁷ M xanthine; however, it decreased in response to 7.5×10⁻⁷ M or higher concentrations. Free radical-induced ODC activity was significantly decreased by addition of the free radical scavengers, SOD, CAT or GPX. In addition, the hypoxic-induced increases in ODC activity and malonaldehyde concentration was also eliminated by the addition of SOD with CAT. Conclusions. (1) Oxygen free radicals, particularly hydroxyl radical (OH^.), appear to trigger an induction of ODC activity in newborn rat cerebrum slices. (2) Oxygen free radicals also appear to mediate the hypoxic-induced increase in ODC activity. (3) Any consequent increase in polyamine synthesis may have profound effects on neurogenesis and neurodifferentiation in the developing brain.     

Immunohistochemical localization of CDK5 activator p39 in the rat brain

We examined the immunohistochemical localization of p39, the regulatory subunit of cyclin-dependent kinase 5 (Cdk5), in the adult rat brain. Cdk5 requires either p39 or p35 to assume its active form in the mammalian CNS. We developed a specific antibody against p39, and localized p39-immunoreactivity in the rat brain. p39-immunoreactivity was observed in the neuronal somata of the cerebral cortex, hippocampus and basal ganglia, and also in the Purkinje cells. Immunopositive astrocytes and oligodendrocytes were scattered throughout the rat brain. p35 immunoactivity has been observed exclusively in neurons and was never detected in glial cells. The present results suggest that p39 and p35 may have different functional roles in the regulation of Cdk5 in the rat CNS.     

Immunohistochemical localization of a melanin concentrating hormone-like peptide in the rat brain

Using antisera generated in rabbits against salmon melanin concentrating hormone (MCH) coupled to human thyroglobulin, the distribution of MCH-like immunoreactivity was mapped throughout the rat central nervous system. The distribution of MCH-like immunoreactivity in rat brain is unique and different from the distribution of other neuropeptides. MCH-like immunoreactive perikarya and fibers are predominant in the posterior hypothalamic area, mostly in the medial forebrain bundle-lateral hypothalamic area subzona incerta and the perifornical area. Cell bodies are located mainly in the medial forebrain bundle and in proximity to well defined hypothalamic nuclei. Fibers are seen throughout the rat brain in all neocortical areas, the neostriatum and the amygdala, in the diencephalon in most hypothalamic nuclei, the habenula, the mamillary body and very dense in the medial forebrain bundle and just ventral to the zona incerta ("subzona incerta"). In the mesencephalon there are fibers in the central gray; in the pons-medulla fibers are contained in the dorsal and ventral parabrachial nuclei; in the tegmental area ventral to the fourth ventricle; in the spinal trigeminal area, the substantia gelatinosa and the reticular nuclei. In the spinal cord there are more fibers in the dorsal than in the ventral horn. The posterior pituitary also contained few MCH-like fibers. It is suggested that a peptide similar, but not identical, to salmon MCH is present in the rat central nervous system.     

Immunohistochemical localization and distribution of torsinA in normal human and rat brain

Dystonia is a disease of basal ganglia function, the pathophysiology of which is poorly understood. Primary torsion dystonia is one of the most severe types of inherited dystonia and can be transmitted in an autosomal dominant manner. Recently, one mutation causing this disorder was localized to a gene on chromosome 9q34, designated DYT1, which encodes for a novel protein termed torsinA. The role of this protein in cellular function, in either normal or dystonic individuals is not known. We have developed a polyclonal antibody to torsinA and report its localization and distribution in normal human and rat brain. We demonstrate that torsinA is widely expressed in brain and peripheral tissues. Immunohistochemical studies of normal human and rat brain reveal the presence of torsinA in the dopaminergic neurons of the substantia nigra pars compacta (SNc), in addition to many other regions, including neocortex, hippocampus, and cerebellum. Labeling is restricted to neurons, as shown by double-immunofluorescence microscopy, and is present in both nuclei and cytoplasm. An ATP-binding property for torsinA has been suggested by its homology to ATP-binding proteins; this was confirmed by enrichment of torsinA in ATP-agarose affinity-purified fractions from tissue homogenates. An understanding of the role of torsinA in cellular function and the impact of the mutation (deletion of a glutamic acid at residue 303) is likely to provide insights into the etiopathogenesis of primary dystonia.     

Immunohistochemical localization of candidates for vesicular glutamate transporters in the rat brain

Vesicular glutamate transporter 1 (VGluT1) is one of the best markers for glutamatergic neurons, because it accumulates transmitter glutamate into synaptic vesicles. Differentiation-associated Na(+)-dependent inorganic phosphate cotransporter (DNPI) shows 82% amino acid identity to VGluT1, and is another candidate for vesicular glutamate transporters. Here, we report the immunocytochemical localization of DNPI and compare it with that of VGluT1 in the adult rat brain. Both DNPI and VGluT1 immunoreactivities were found mostly in neuropil, presumably in axon terminals, throughout the brain. In the telencephalic regions, intense DNPI immunoreactivity was observed in the glomeruli of the olfactory bulb, layer IV of the neocortex, granular layer of the dentate gyrus, presubiculum, and postsubiculum. In contrast, VGluT1 immunoreactivity was intense in the olfactory tubercle, layers I-III of the neocortex, piriform cortex, entorhinal cortex, hippocampus, dentate gyrus, and subiculum. In the thalamic nuclei, DNPI-immunoreactive terminal-like profiles were much larger than VGluT1-immunoreactive ones, suggesting that DNPI immunoreactivity was subcortical in origin. DNPI immunoreactivity was much more intense than VGluT1 immunoreactivity in many brainstem and spinal cord regions, except the pontine nuclei, interpeduncular nucleus, cochlear nuclei, and external cuneate nucleus. In the molecular layer of the cerebellar cortex, climbing-like fibers showed intense DNPI immunoreactivity, whereas neuropil contained dense VGluT1-immnoreactive deposits. Both DNPI and VGluT1 immunoreactivities were observed as mossy fiber terminal-like profiles in the cerebellar granular layer. DNPI and VGluT1 immunoreactivities appeared associated with synaptic vesicles in the axon terminals forming asymmetric synapses in several regions examined electron microscopically. The present results indicate that DNPI and VGluT1 are used by different neural components in most, if not all, brain regions, suggesting the complementary functions of DNPI and VGluT1.     

Immunohistochemical localization of a GHB receptor-like protein isolated from rat brain

Gamma-hydroxybutyrate (GHB) is a substance derived from the metabolism of GABA and is heterogeneously distributed in various regions of the brain. This compound possesses a neuromodulatory role on several types of synapses, particularly those using GABA as a neurotransmitter. At physiological concentrations, this effect of GHB is mediated via specific receptors that induce neuronal hyperpolarization and bind radioactive GHB with a specific distribution, ontogenesis, kinetics, and pharmacology. A membrane protein that possesses six to seven transmembrane domains and which binds and is activated by micromolar amounts of GHB was recently cloned from rat brain hippocampus. In order to study the regional and cellular distribution of this receptor in rat brain, we selected several specific peptides belonging to the extracellular domains of the receptor to be used as specific immunogens to raise polyclonal antibodies in the rabbit. Among the antisera obtained, one of them gave particularly good results in terms of specificity and reactivity at high dilution. Immunohistochemical analyses, both at the confocal and electron microscopic level, showed receptor protein distribution closely resembling the distribution of GHB high-affinity binding sites, except for cerebellum, where GHB receptor(s) of lower affinity exist(s). In all regions studied the GHB receptor-like protein labeling appears to be distributed specifically in neurons and not in glial cells. At the cellular level the antibody specifically labels dendrites, and no immunoreactivity was detected in presynaptic endings or in axons. Accordingly, electron microscopy reveals strong labeling of postsynaptic densities and of neuronal cytosol.     

Immunohistochemical localization of enkephalin in rat forebrain

Discrete localization of enkephalin immunoreactive sites in the rat forebrain was undertaken using animals with and without colchicine pretreatment. Both the immunofluorescence and unlabeled PAP antibody techniques were employed using antisera directed against methionine-enkephalin. We have found several areas of enkephalin fiber and cell localizations not previously described, as well as confirming the results of other researchers. Thus, wer found large amounts of perikaryal enkephalin immunoreactivity in the lateral septal nucleus, bed nucleus of the stria terminalis, the preoptic nuclei, the ventral premammillary nucleus, prelateral mammillary nucleus, the medial mammillary nucleus, paraventricular nucleus, the periventricular, ventromedial, dorsomedial and posterior nuclei of the hypothalamus, the arcuate nucleus, and the ventral nucleus of the lateral geniculate body.High concentrations of immunoreactive neuronal fibers not immediately associated with enkephalin perikarya were seen in the nucleus accumbens, medial fore-brain bundle, globus pallidus, mammillothalamic tract, the subthalamic nucleus, and the caudal portion of the zona incerta. Many of the areas presented in this study correspond with areas known to exert some regulation over neuroendocrine function as well as various motor and sensory functions. Nuclei with high immunoreactive fiber content also correspond in many instances with areas reported to have high concentrations of opiate receptors. Possible enkephalin-containing tracts and the relationships of enkephalin-containing cells in fiber bundles are also pointed out.     

Radiohistochemical localization of insulin receptors in the adult and developing rat brain

The light microscopic localization of insulin receptors in the rat central nervous system has been investigated by means of a new autoradiographic technique. These receptors are fairly evenly distributed in the adult brain except in the olfactory bulb. There, the external plexiform layer is especially rich in them. Insulin binding sites were more heterogeneously distributed in the fetal brain and exhibited a waxing and waning during development. Highest densities were observed at 15 days of gestation.     

Immunohistochemical localization and biochemical characterization of nerve growth factor receptor in adult rat brain

The expression of nerve growth factor (NGF) receptor in adult rat brain was studied by immunohistochemistry with a specific anti-rat NGF receptor monoclonal antibody, 192-IgG. Intense NGF receptor immunoreactivity (NGFRI) was found in structures known to be NGF responsive, including forebrain cholinergic neurons in medial septum, diagonal band of Broca, and basal nucleus of Meynert; central processes of neural-crest-derived sensory ganglion neurons and their innervated nucleus also contained such immunoreactivity. Distinct NGFRI staining was also found in many brain areas and cell types not known to be NGF responsive, including some hypothalamic regions, circumventricular organs, some areas related to the optic system, olfactory glomeruli, ependymal and subependymal cells in some locations, mesencephalic nucleus of the trigeminal nerve, cerebellar molecular layer, central linear nucleus, solitary tract and its nucleus, and inferior olive. The NGFRI in the circumventricular organs was further studied by in vivo labeling of 125I-ligands. Intravenously injected 125I-NGF, but not 125I-cytochrome c, was specifically accumulated in the area postrema. Biochemical study of the NGF receptor showed a major band of molecular weight of approximately 90 KDa in the area postrema, choroid plexus, median eminence, and medial septum with the relative content consistent with that seen by immunohistochemistry. No evidence of a truncated NGF receptor was observed. The results of this study suggest that NGF and its receptor have broader roles in adult mammalian brain than previously thought.     

Ornithine decarboxylase and polyamines in developing rat brain and heart: Effects of perinatal hypothyroidism

Hypothyroidism induced by perinatal administration of propylthiouracil (PTU) had profound effects on growth of the heart, with major organ weight deficits persisting well beyond the termination of drug treatment. These effects were preceded by disruption of the developmental patterns of cardiac ornithine decarboxylase (ODC) and the polyamines, which are thought to be intracellular modulators of cellular maturation. Activity of cardiac ODC was depressed in the PTU-treated group and putrescine and spermidine levels were markedly subnormal. PTU administration also affected brain growth, but much less so than in the heart. The disruption of the brain ODC/polyamine system was also less pronounced, with relatively small degrees of spermidine depletion and a slight elevation of ODC. For both tissues, the biochemical effects of perinatal hypothyroidism were opposite to those found previously for administration of exogenous thyroid hormones. These results support the views that:

1. (1) endogenous thyroid hormones contribute to the maintenance of normal developmental patterns of ODC and the polyamines, and

2. (2) the ODC/polyamine system participates in events modulating subsequent tissue growth.