Self-injurious behavior: gene-brain-behavior relationships.

This paper summarizes a conference held at the National Institute of Child Health and Human Development on December 6-7, 1999, on self-injurious behavior [SIB] in developmental disabilities. Twenty-six of the top researchers in the U.S. from this field representing 13 different disciplines discussed environmental mechanisms, epidemiology, behavioral and pharmacological intervention strategies, neurochemical substrates, genetic syndromes in which SIB is a prominent behavioral phenotype, neurobiological and neurodevelopmental factors affecting SIB in humans as well as a variety of animal models of SIB. Findings over the last decade, especially new discoveries since 1995, were emphasized. SIB is a rapidly growing area of scientific interest to both basic and applied researchers. In many respects it is a model for the study of gene-brain-behavior relationships in developmental disabilities.

Administration of unmodified prolactin (U-PRL) and a molecular mimic of phosphorylated prolactin (PP-PRL) during rat pregnancy provides evidence that the U-PRL:PP-PRL ratio is crucial to the normal development of pup tissues.

During rat pregnancy initial high concentrations of prolactin (PRL) decline by about day 9, concomitant with an increase in the ratio of unmodified to phosphorylated PRL. The physiological significance of both the decline in total PRL and the change in ratio of the two PRLs is unknown. To test the importance of each, either unmodified PRL (U-PRL) or a molecular mimic of phosphorylated PRL (PP-PRL) were continuously administered to rats throughout pregnancy. A dose of 6 microg/24 h resulted in circulating concentrations of 50 ng/ml of each administered PRL and had little effect on the pregnancy itself. After birth, pups were killed and various tissues examined. In the pup lungs, exposure to additional PP-PRL caused a reduction in epithelial integrity and an increase in apoptosis, whereas exposure to additional U-PRL had beneficial, anti-apoptotic effects. In the heart, PP-PRL caused an apparent developmental delay, whereas U-PRL promoted tissue compaction. In the blood, U-PRL increased the number of mature red blood cells at the expense of white blood cell production. Within the white blood cell population, myelopoiesis was favored at the expense of lymphopoiesis. PP-PRL, in contrast, had a less dramatic influence on the hematopoietic compartment by promoting red blood cell maturation and granulocyte production. In the thymus, exposure to PP-PRL caused accumulation of apoptotic thymocytes in enlarged glands, whereas exposure to U-PRL resulted in smaller thymi. In the spleen, exposure to U-PRL increased cellularity, with the majority of cells belonging to the erythroid series - a finding consistent with increased red blood cells in the circulation. Exposure to PP-PRL was without discernible effect. In all of these tissues, the contrasting effects of the two PRLs indicate that the absolute concentration of PRL is not crucial, but that the ratio of U-PRL to PP-PRL has a profound effect on tissue development. In brown fat, both PRL preparations decreased the number of lipid droplets. This result is therefore probably a consequence of the increase in total PRL. The results of this study attest to the importance of the U-PRL:PP-PRL ratio normally present during pregnancy and have provided clues as to the possible pathogenesis of a variety of neonatal problems.

Mouse molecular cytogenetic resource: 157 BACs link the chromosomal and genetic maps.

We have established a collection of strong molecular cytogenetic markers that span the mouse autosomes and X chromosome at an average spacing of one per 19 Mb and identify 127 distinct band landmarks. In addition, this Mouse Molecular Cytogenetic Resource relates the ends of the genetic maps to their chromosomal locations. The resource consists of 157 bacterial artificial chromosome (BAC) clones, each of which identifies specific mouse chromosome bands or band borders, and 42 of which are linked to genetic markers that define the centromeric and telomeric ends of the Whitehead/MIT recombinational maps. In addition, 108 randomly selected and 6 STS-linked BACs have been assigned to single chromosome bands. We have also developed a high-resolution fluorescent reverse-banding technique for mouse chromosomes that allows simultaneous localization of probes by fluorescence in situ hybridization (FISH) with respect to the cytogenetic landmarks. This approach integrates studies of the entire mouse genome. Moreover, these reagents will simplify gene mapping and analyses of genomic fragments in fetal and adult mouse models. As shown with the MMU16 telomeric marker for the trisomy 16 mouse model of Down syndrome, these clones can obviate the need for metaphase analyses. The potential contribution of this resource and associated methods extends well beyond mapping and includes clues to understanding mouse chromosomes and their rearrangements in cancers and evolution. Finally it will facilitate the development of an integrated view of the mouse genome by providing anchor points from the genetic to the cytogenetic and functional maps of the mouse as we attempt to understand mutations, their biological consequences, and gene function.

Impairments in learning and memory accompanied by neurodegeneration in mice transgenic for the carboxyl-terminus of the amyloid precursor protein.

In Alzheimer's disease (AD), a progressive decline of cognitive functions is accompanied by neuropathology that includes the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature. We have proposed that a fragment of the Alzheimer amyloid precursor protein (APP) comprising the carboxyl-terminal 100 amino acids of this molecule (APP-C100) plays a crucial role in the neurodegeneration and subsequent cognitive decline in AD. To test this hypothesis, we performed behavioral analyses on transgenic mice expressing APP-C100 in the brain. The results revealed that homozygous APP-C100 transgenic mice were significantly impaired in cued, spatial and reversal performance of a Morris water maze task, that the degree of the impairment in the spatial learning was age-dependent, and that the homozygous mice displayed significantly more degeneration of neurons in Ammon's horn of the hippocampal formation than did heterozygous or control mice. Among the heterozygotes, females were relatively more impaired in their spatial learning than were males. These findings show that expression of APP-C100 in the brain can cause age-dependent cognitive impairments that are accompanied by hippocampal degeneration.

Characterization of N-methyl-d-aspartate receptors in the hyperammonemic sparse fur mouse.

The N-methyl-d-aspartate (NMDA) receptor, a glutamate receptor subtype, is a ligand-gated ion channel. Overstimulation of NMDA receptors may increase intracellular Ca2+ concentrations to lethal levels in neurodegenerative disorders affecting the basal ganglia. Such excitotoxicity may also contribute to the loss of medium spiny neurons in the striata of the hyperammonemic sparse fur (spf/Y) mouse, a model of the X-linked disorder of the urea cycle, ornithine carbamoyltransferase deficiency (OCTD). Levels of quinolinic acid (QA), a potent NMDA agonist, are elevated in the brains of spf/Y mice. Further, direct injection of QA into the striatum produces selective degeneration of medium spiny neurons. Microglia, an endogenous source of QA in the brain, are abundant in spf/Y mice during the period of neuronal degeneration. The location and density of NMDA receptors was visualized by gold labelled immunocytochemistry with a polyclonal antibody to the NMDAR1 receptor subtype and their distribution quantified. A 58% reduction was found in the median density value in the layer V pyramidal neurons in fronto-parietal cortex (p<0.001), but no significant change was observed in the striatum. NMDA receptor binding was examined using [3H]dizocilpine ([3H]MK-801). Receptor density (Bmax) in the striata of clinically stable spf/Y mice and +/Y littermates was unchanged, but was decreased 15% (p<0.01) in the fronto-parietal cortices in clinically stable spf/Y mice compared with +/Y littermate controls.

Dendritic alterations in cortical pyramidal cells in the sparse fur mouse.

Ornithine carbamoyltransferase deficiency, an X-linked trait, leads to toxic hyperammonemia in sparse fur (spf/Y) mice. Quantitative analysis of the basilar dendritic tree of layer V pyramidal cells in frontoparietal cortex stained by the Golgi Kopsch method revealed a significant decrease in both the complexity of the dendritic arbor and in dendritic terminal spine density (60%) in spf/Y mice compared with controls. Such reductions may contribute to behavioral dysfunction observed in spf/Y mice.

Age-dependent neuronal and synaptic degeneration in mice transgenic for the C terminus of the amyloid precursor protein.

The molecular basis for the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature in Alzheimer's disease (AD) is incompletely understood. We have proposed that one molecule common to these abnormal processes is a fragment of the Alzheimer amyloid precursor protein (APP) comprising the C-terminal 100 amino acids of this molecule (APP-C100). We tested this hypothesis by creating transgenic mice expressing APP-C100 in the brain. We report here that aging (18-28 month) APP-C100 transgenic mice exhibit profound degeneration of neurons and synapses in Ammon's horn and the dentate gyrus of the hippocampal formation. Of the 106 transgenic mice between 8 and 28 months of age that were examined, all of those older than 18 months displayed severe hippocampal degeneration. The numerous degenerating axonal profiles contained increased numbers of neurofilaments, whorls of membrane, and accumulations of debris resembling secondary lysosomes near the cell body. The dendrites of degenerating granule and pyramidal cells contained disorganized, wavy microtubules. Cerebral blood vessels had thickened refractile basal laminae, and microglia laden with debris lay adjacent to larger venous vessels. Mice transgenic for Flag-APP-C100 (in which the hydrophilic Flag tag was fused to the N terminus of APP-C100) showed a similar degree of neurodegeneration in the hippocampal formation as early as 12 months of age. The 45 control mice displayed only occasional necrotic cells and no extensive cell degeneration in the same brain regions. These findings show that APP-C100 is capable of causing some of the neuropathological features of AD.

Transgenic mice expressing APP-C100 in the brain.

The classic hallmarks of Alzheimer's disease are the deposition of amyloid in plaques and in the cerebrovasculature, and the emergence of neurofibrillary tangles in neurons. The interplay between these two pathologic processes, on the one hand, and the degeneration of neurons and loss of cognitive functions on the other, remains incompletely understood. We have proposed that one crucial component of this interplay is a fragment of the Alzheimer amyloid protein precursor (APP) comprising the carboxyterminal 100 amino acids of this molecule, which we term APP-C100 (or, more simply, C100). This fragment, which comprises the 42-amino acid amyloid protein (A beta) and an additional 58 amino acids carboxyterminal to it, was found to be toxic specifically to nerve cells in vitro. We developed transgenic mouse models to test the hypothesis that APP-C100 causes Alzheimer's disease neuropathology. APP-C100 was delivered to the mouse brain via a transgene expressing C100 under the control of the dystrophin brain promoter. These transgenic animal models for the action of APP-C100 in the brain exhibited some of the neuropathological features characteristic of Alzheimer disease brain. The animal models that we have created can be used to test hypotheses concerning the mechanism by which C100 interacts with a neuronal receptor to kill neurons.

Evidence of excitotoxicity in the brain of the ornithine carbamoyltransferase deficient sparse fur mouse.

Ornithine carbamoyltransferase deficiency (OCTD) is the most common inborn error of urea synthesis. An X-linked disorder, OCTD males commonly present with hyperammonemic coma in the newborn period. There is a high rate of mortality and morbidity, with most survivors sustaining severe brain damage and resultant developmental disabilities. Although ammonia is presumed to be the principal neurotoxin, there is evidence that other neurochemical alterations may also be involved. The OCTD sparse fur (spf/Y) mouse has proven to be a useful model of this disease with similar metabolic and neurochemical alterations to those found in the human disease. In this study, the levels of the tryptophan derived excitotoxin quinolinic acid were examined in the brains of spf/Y mice. In addition, the neuropathology was examined using both light and electron microscopic approaches. Consistent with reports in children with urea cycle disorders, the levels of tryptophan and quinolinic acid were increased two-fold in various brain regions of the spf/Y mouse. Quinolinic acid, an agonist at the N-methyl-D-aspartate (NMDA) receptors, is known to produce selective cell loss in the striatum. We found a significant loss of medium spiny neurons and increased numbers of reactive oligodendroglia and microglia in the striatum of spf/Y mice. These neurochemical and neuropathological observations are consistent with an excitotoxic influence on brain injury in OCTD. It leads us to suggest that administration of NMDA receptor antagonists may ameliorate brain damage in children with inborn errors of urea synthesis.

The neuropathology of the trisomy 16 mouse.

The purpose of this review is to describe the neuropathology of mouse trisomy 16 and to compare that pathology with the pathologic features observed in individuals with Down syndrome (DS) trisomy 21 (Ts21). Additionally, we will compare the neuroanatomic, neurochemical, and neurophysiologic abnormalities observed in both DS and in mouse trisomy 16 (Ts16). We discuss strategies that have been used to circumvent the failure of trisomy 16 mice to survive into the postnatal period: the creation of chimeras, the use of transplantation of fetal trisomic nervous tissue into normal hosts, and the generation of mice with partial Ts16. We compare the results with those observed in DS and in fetal mouse Ts16. Like individuals with DS, mice with trisomy 16 have triplication of a constellation of genes that have remained together in the same order for roughly 80 million years. As more of these genes have been identified and localized to specific regions of human chromosome 21 and mouse chromosome 16, efforts have been made to create transgenic mice that carry single of these genes in excess. In this review, we also discuss the comparative pathology observed in these transgenic mice.

Abnormal production of interleukin-1 by microglia from trisomy 16 mice.

The production of interleukin-1 (IL-1) was examined in cultured CNS microglia obtained from trisomy 16 (Ts16) fetal mouse brain, a model system for studies relevant to Down syndrome (DS). When compared to microglia from their normal littermates, Ts16 microglia produced significantly higher levels of IL-1 activity both before and following stimulation with lipopolysaccharide (LPS). IL-1 release was stimulated by alpha/beta interferon (IFN) in the normal but not Ts16 microglial cultures. The overall level of IL-1 production in normal littermates, however, was still less than that seen in Ts16. Thus, microglia from Ts16 mice may function in an inappropriate manner and, if this abnormality occurs in vivo, may have wide ranging effects on a developing nervous system.

Developmental expression of the gene encoding growth-associated protein 43 (Gap43) in the brains of normal and aneuploid mice.

The gene encoding growth-associated protein 43 (Gap43), a neuronal phosphoprotein associated with axonal outgrowth and synaptic plasticity, is located on mouse chromosome 16 (MMU16). We examined the developmental expression of Gap43 in normal, trisomy 16 (Ts16), and trisomy 19 (Ts19) mouse brain using northern blot analysis and in situ hybridization as a first step toward understanding the neurobiologic consequences of increased gene dosage on brain development. Gap43 expression was detected by in situ hybridization throughout the mesencephalon, rhombencephalon, spinal cord, and first branchial arch in whole embryos as early as day 10 of gestation (E10). By E15, Gap43 expression was localized to cells in the retina, the olfactory bulbs, and anterior olfactory structures, the cortical plate, the basal telencephalon, diencephalon, midbrain, hindbrain, and spinal cord. Northern blot analysis detected a three-fold increase in Gap43 mRNA levels in the brains of normal mice between E12-E18. At E15, Gap43 mRNA levels were increased 35-40% in Ts16 mouse brain and decreased 10% in Ts19 mouse brain, relative to euploid littermate controls. Using in situ hybridization we found that overexpression of Gap43 occurred in the diencephalon, medial and lateral basal telencephalon, and cortical plate region in Ts16 mice relative to littermate controls. Thus, the degree of overexpression of Gap43 mRNA in Ts16 mice is consistent with that expected from gene dosage effects.

Newborn basal forebrain lesions disrupt cortical cytodifferentiation as visualized by rapid Golgi staining.

We have previously shown that neonatal lesions of the basal forebrain cholinergic afferents result in transient cholinergic depletion concomitant with abnormal morphogenesis of cerebral cortex in Balb/CByJ mice (Höhmann et al., 1988). Here, we have utilized the rapid Golgi method to further characterize these previously observed abnormalities. We compared layer V pyramidal neurons in somatomotor cortex ipsi- and contralateral to the lesion at postnatal days (PND) 7 and 14. Quantitative evaluations showed a significant reduction in all aspects of the dendritic tree as well as in cell body size in ipsilateral cortex at PND 7. Differences between ipsi- and contralateral pyramidal cells had attenuated by PND 14, but significant somatic size differences persisted, as did changes in the apical branching pattern. Qualitative differences between ipsilateral and contralateral hemispheres included the relatively more immature appearance of ipsilateral neurons at both ages, in addition to unusual dendritic morphology, particularly at PND 14. A close correlation was apparent between the magnitude of cholinergic depletion in cortex (larger at PND 7 than at PND 14) and the severity of abnormalities in pyramidal cell morphogenesis. We conclude that a normal cholinergic innervation to neocortex is instrumental in the timely differentiation of cortical neurons, because neonatal nBM lesions disrupted the time schedule of differentiation, but did not preclude the pyramidal neurons from further differentiation at a later time.

Expression of the amyloid precursor protein gene in mouse oocytes and embryos.

The amyloid precursor protein (APP) is thought to be processed aberrantly to yield the major constituent of the amyloid plaques observed in the brains of patients with Alzheimer disease and Down syndrome. However, the gene encoding APP is expressed widely in normal human tissues and in adult and fetal mouse tissues and is alternatively spliced in a tissue-specific pattern in the adult. There is evidence that APP may function as a growth factor and as a mediator of cell adhesion and in these roles could be important in morphogenesis. As a step toward determining the role of APP in development and in determining how the adult pattern of tissue-specific splicing is established, we have used reverse transcription and the polymerase chain reaction to demonstrate APP expression in mouse oocytes, preimplantation embryos, and postimplantation embryonic stages to the late embryonic period. All three splicing forms described in mouse were present at each stage, although there were changes in the ratios of the splicing forms at different stages. Screens for APP clones in embryonic cDNA libraries from the egg cylinder stage and the early somite stage were used to confirm the results of the polymerase chain reaction, and APP clone abundance was found to increase 10-fold between the two stages.

Enhanced production of superoxide anion by microglia from trisomy 16 mice.

Disruption of normal oxygen radical metabolism in the CNS may contribute to the neuropathological changes associated with Down syndrome (trisomy 21) and its mouse counterpart, the trisomy 16 (Ts16) mouse. One potent source of oxyradicals is the CNS-specific macrophage, the microglial cell. We prepared primary glial cultures from the cerebral cortices of Ts16 and normal littermate mice taken at day 15 of gestation. Microglia were isolated from confluent cultures after 14 days in vitro and assayed for superoxide anion production using a cytochrome C reduction assay. Stimulation by either opsonized zymosan (OPZ) or phorbol myristate acetate (PMA), produced significantly higher levels (2.8-20 fold) of superoxide per mg protein in Ts16 microglial cultures. Resting, i.e. unstimulated secretion, was not significantly different from littermate controls. Astrocyte enriched cultures, stimulated by OPZ, exhibited low levels of superoxide production which was higher in Ts16 mice than normal littermates. Microglial enriched cultures from rat neonatal cerebral cortices were exposed for 24 h to medium from the Ts16 glial cultures. Superoxide production in the Ts16 media treated rat microglia was significantly higher than in those treated with littermate conditioned media.

Developmental expression of somatostatin in mouse brain. II. In situ hybridization.

The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.

Increased number of somatostatin-immunoreactive neurons in primary cultures of trisomy 16 mouse neocortex.

The gene encoding for pre-prosomatostatin is located on chromosome 16 of the mouse. To determine the effect of an extra copy of this gene on somatostatin expression in neurons, primary disaggregated cultures of neocortex prepared from 15 days gestational Trisomy 16 mice and their littermate euploid controls were subjected to immunocytochemical staining for somatostatin, neuropeptide Y and glutamic acid decarboxylase. The results demonstrate a selective and significant increase in the number of somatostatin-immunoreactive neurons.