Delayed cell migration in the developing rat brain following maternal omega 3 alpha linolenic acid dietary deficiency.

Diminished levels of docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid (FA) synthesized from alpha linolenic acid (ALA, 18:3n-3), have been implicated in changes in neurotransmitter production, ion channel disruption and impairments of a variety of cognitive, behavioral and motor functions in the perinatal and adult mammal. Neuronal migration in the cortex and hippocampus of newborn and postnatal rats after ALA-deficiency, beginning on the 2nd day after conception and continuing for three weeks, was investigated. A marked decrease in the migration of bromodeoxyuridine((+))/neuronal nuclei((+))/neurofilament((+)) and glia fibrillary acidic protein((-)) neuronal cells to the dense cortical plate was accompanied by a corresponding abundance of non-migrating cells in several regions such as cortical layers IV-VI, corpus callosum and the sub-ventricular zone of ALA-deficient newborns. Similarly, a delayed migration of cells to CA1 and dentate gyrus areas was noticed while most cells were retained in the subicular area adjacent to the hippocampus. The reversibility of delay in migration in the hippocampus and cortex, after one and two weeks respectively, may be attributed to a temporary reelin disorganization or partial deficiency. Transient obstruction of neuronal cell migration may have long-lasting consequences on the organization of neuronal assemblies, on the connection between neurons (lateral connections) and acquisition of function in the adult brain.

Neuroprotective effect of rasagiline, a monoamine oxidase-B inhibitor, on spontaneous cell degeneration in a rat model.

Spontaneously hypertensive rats (SHR) pathologically elevate blood pressure with age. This elevation is accompanied by specific neuronal degeneration in the hypothalamus and enlargement of the lateral ventricles. The aim of this study was to assess the neuroprotective effect of the monoamine oxidase B (MAO-B) inhibitor, rasagiline on paraventricular (PVN) hypothalamic degeneration in SHR. The S-enantiomer of rasagiline, S-PAI, a much weaker MAO inhibitor, and two antihypertensive drugs, captopril and hydralazine were also tested. Normotensive Wistar Kyoto (WKY) rats served as controls. One month-old SHR or WKY rats were treated daily for 3-4 months. Systolic blood pressure was recorded, parvocellular vasopressin (VP) immunopositive cells were counted and the area of the third ventricle measured. In saline-treated SHR, blood pressure rose significantly and the number of VP parvocellular cells was reduced by about 60% relative to WKY. Rasagiline, 1 mg/kg/day, reduced PVN neuronal cell death in SHR up to 112% relative to saline-treated SHR; 0.3 mg/kg/day exerted a smaller but significant effect. These actions were accompanied by parallel reductions in systolic blood pressure. Captoril, hydralazine and S-PAI did not prevent death of VP neurons. In SHR, the volume of the third ventricle was about double that of WKY. Rasagiline significantly prevented this ventricular dilation. These results indicate than rasagiline protects from cell death in an in vivo animal model in a dose-dependant manner and could be of use as a neuroprotector in the central nervous system.

Late degeneration of nigro-striatal neurons in ATM-/- mice.

The generation of an Atm -/- mouse model of the human ataxia-telangiectasia (AT) opened new avenues toward a better understanding of the molecular and cellular basis of AT. We have recently reported that 5-month-old Atm-/- mice exhibit severe loss of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, down to 26% of age-matched controls. In the present study we analyzed development of the dopaminergic cell loss in the context of the nigro-striatal system. We found that dopaminergic neurons are formed normally in the Atm-/- mouse, and degenerate during the first few months of life; there was no difference between 1-month-old Atm-/- and control mice in the number of dopaminergic cells that were retrogradely labeled by an injection of fluorescent tracer into the striatum. On the other hand, a dramatic reduction in the number of labeled cells was found in 5-month-old Atm-/- mice. This cell loss was significant in areas A9 and A10 but not in area A9-I. These findings indicate that midbrain dopaminergic neurons in Atm-/- mice initially send normal axons to the striatum, only to degenerate later in life. In addition, an age-dependent as well as topographic, medial-to-lateral loss of GAD, met-enkephaline and substance-P immunopositive cells was found in the striatum of the Atm-/- mice. This phenomenon was significant only in the 5-month-old Atm-/- mice (3 months after the beginning of detectable dopaminergic cell loss). In both the striatum and the substantia nigra, the apparent cell loss was accompanied by gliosis. In addition, alpha-synuclein immunopositive bodies were observed in the cortex, striatum and substantia nigra of these mice. The present data indicate that Atm-/- mice exhibit a progressive, age-dependent, reduction in dopaminergic cells of the substantia nigra, followed by a reduction in projection neurons of the striatum. Thus, the Atm-/- mouse may model the extrapyramidal motor deficits seen in AT patients.

Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis.

The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

Spontaneous hyperplasia of the ventral lobe of the prostate in aging genetically hypertensive rats.

Recent studies have shown that the prostatic autonomic innervation takes part in its homeostasis and growth. Other works showed that spontaneously hypertensive rats (SHR) show excessive sympathetic activity, accompanied by lower urinary tract symptoms, increased growth capacity of prostatic stromal cells, and increased levels of androgens and their receptors. Furthermore, young SHR were reported to present incipient stages of benign prostatic hyperplasia (BPH). The aim of the present study was to examine whether this strain indeed develops spontaneous BPH with age, and can thus serve as a genuine natural model for this disorder. For this purpose, ventral lobes of prostates of one-year-old, male SHR and their normotensive counterparts, Wistar Kyoto (WKY) rats, were examined histopathologically, and the degree of hyperplasia was evaluated according to a score-chart protocol (histoscore). SHR exhibited severe adenomatous spontaneous BPH, characterized by piling-up of epithelial cells, with papillary formations, accompanied by a mild increase in the amount of fibrocytes and smooth muscle cells in the stroma. This was reflected by histoscore values of 38 +/-2. Thickening of prostatic arterioles also was noted, as well as mild chronic inflammatory exudate. WKY rats did not show any of these features of BPH despite their age (histoscore 17 +/- 3, significantly different from that of SHR). We conclude that SHR can serve as a rodent model for the spontaneous development of BPH with age, most probably due to the excessive neuroendocrine activity characteristic of this rat strain.

Locomotor activity causes a rapid up-regulation of vasoactive intestinal peptide in the rat hippocampus.

Vasoactive intestinal peptide (VIP) expression is restricted to interneurons in the hippocampus of normal adult rats. However, 3-6 hours after a 60-minute walk in an activity wheel, VIP was transiently expressed in most pyramidal and granular neurons of the hippocampus. Locomotion was also associated with a dramatic increase in VIP immunoreactivity in the motor cortex, primarily in bipolar cells. Reverse transcriptase-polymerase chain reaction analysis indicated that VIP mRNA increases transiently by more than twofold, before the increases in peptide immunoreactivity in both the hippocampus and motor cortex. By comparison, another marker of inhibitory interneurons, glutamate decarboxylase, did not change its expression pattern after locomotion. The calcium binding protein, calbindin-D28K, normally expressed in interneurons, was now found also in glial cells of the hippocampus and motor cortex. Another marker of enhanced electrical activity, the immediate early gene, c-Fos, was expressed in pyramidal and granular neurons at 3 hours but not at 6 hours after locomotion. These results suggest that mapping of peptide expression in the brain of a docile, inactive rat may not reflect the real distribution and functions of a peptide in an active animal.

Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice.

Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor L-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug D-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.

Activity-dependent regulation of Neu differentiation factor/neuregulin expression in rat brain.

Neu differentiation factor (NDF/neuregulin) is widely expressed in the central and peripheral nervous systems, where it functions as a mediator of the interactions between nerve cells and Schwann, glia, oligodendrocyte, and muscle cells, to control cellular proliferation, differentiation, and migration. NDF binds to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Here we demonstrate that NDF and its ErbB-4 receptor are highly reactive to changes in ambient neuronal activity in the rodent brain in a region-selective manner. Generation of epileptic seizures by using kainic acid, a potent glutamate analog, elevated levels of NDF transcripts in limbic cortical areas, hippocampus, and amygdala. Concomitantly, ErbB-4 mRNA was increased with a similar spatial distribution, but transcription of the other NDF receptor, ErbB-3, did not change. A more moderate stimulation, forced locomotion, was accompanied by an increase in NDF transcripts and protein in the hippocampus and in the motor cortex. Similar changes were found with ErbB-4, but not ErbB-3. Last, a pathway-specific tetanic stimulation of the perforant path, which produced long-term potentiation, was followed by induction of NDF expression in the ipsilateral dentate gyrus and CA3 area of the hippocampus. Taken together, these results indicate that NDF is regulated by physiological activity and may play a role in neural plasticity.

Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation.

Two receptor tyrosine kinases, ErB-3 and ErbB-4, mediate signaling by Neu differentiation factors (NDFs, also called neuregulins), while ErbB-1 and ErbB-2 serve as co-receptors. We show that the two NDF/neuregulin receptors differ in spatial and temporal expression patterns: The kinase-defective receptor, ErbB-3, is expressed primarily in epithelial layers of various organs, in the peripheral nervous system, and in adult brain, whereas ErbB-4 is restricted to the developing central nervous system and to the embryonic heart. An example of alternating expression of the two receptors is provided by the developing cerebellum: During postnatal cerebellar development, ErbB-4 expression slightly decreases along with a decline in NDF transcription, whereas ErbB-3 expression commences after the peak of neurogenesis. To study functional differences, we established primary brain cultures and found that ErbB-3 was expressed only in oligodendrocytes, whereas ErbB-4 expression was shared by oligodendrocytes, astrocytes and neurons. Blocking the action of endogenous NDF in vitro, by using a soluble form of ErbB-4, accelerated neurite outgrowth in both primary cultures and in neuronal-type cultures of the P19 teratocarcinoma, suggesting an inhibitory effect of NDF on neural differentiation. Apparently, ErbB-3 is associated with proliferation of P19 cells, whereas ErbB-4 correlates with a differentiated phenotype. We conclude that the two NDF receptors play distinct, rather than redundant, developmental and physiological roles.

Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.

Selective elimination of hypothalamic neurons by grafted hypertension-inducing neural tissue.

Embryonic hypothalamic tissue originating from spontaneously hypertensive rats (SHR) was implanted in young normotensive Wistar Kyoto rats in an attempt to localize hypothalamic regions directly responsible for the induction of hypertension. A 25% increase in host systolic blood pressure as compared with the controls was recorded 3 months after implantation in the animals receiving rostral hypothalamic tissue (R-SHR), whereas blood pressure was not affected in the animals grafted with caudal hypothalamic tissue (C-SHR). The hypertension in the R-SHR group was accompanied by hypertrophy of the heart and kidneys. The number of vasopressin-immunopositive (VPi) parvocellular cells in the hypothalamic paraventricular nucleus (PVN) of the R-SHR group was massively reduced (by 72%), while that of the tyrosine hydroxylase-immunopositive cells displayed no change. In the suprachiasmatic nucleus of these animals the VPi cell number was unaltered. In the C-SHR, the amount of parvocellular VPi cells was also unaltered. Likewise, oxytocin-containing cells were the same in all groups. DNA nick-end labeling of the tissue revealed that PVN cells are undergoing programmed cell death. These results implicate a selective degeneration by hypothalamic PVN cells in the pathogenesis of hypertension.

Quantitation of ventricular size in normal and spontaneously hypertensive rats by magnetic resonance imaging.

Magnetic resonance imaging (MRI) performed at high field (4.7 Tesla), and high spatial resolution (0.6 mm slice thickness, 0.18 mm inplane) enabled noninvasive quantitative measurement of the ventricular vol. in live rats. Comparing the results for 15 male Wistar-Kyoto (WKY) rats, aged 2.5-10 months, with those from 17 spontaneously hypertensive rats (SHR), clearly confirmed the previously reported elevated ventricular vols. in the SHR strain. A significant difference in ventricular vol. between the two strains was detected above the age of 3 months. For mature animals above the age of 6 months the mean vol. in the SHR strain was elevated by about a factor of two compared to the WKY control animals.

Hypertension induced by hypothalamic transplantation from genetically hypertensive to normotensive rats.

The role of the hypothalamus (HTH) in the pathogenesis of genetic hypertension was studied in spontaneously hypertensive rats (SHR). It is currently believed that, in this strain, the genetic defect manifests itself mainly in the HTH. We examined this hypothesis by grafting HTH neurons from embryos of SHR or control Wistar Kyoto (WKY) rats into the HTH of adult normotensive WKY rats. Changes in host systolic blood pressure (SBP) were monitored, and alterations in vasoactive intestinal polypeptide (VIP) gene expression of the host brain were studied. In rats grafted with HTH tissue from SHR embryos (G-SHR), the blood pressure rose by 31% as compared with that in the grafted control group. The blood pressure climbed gradually over a period of 6 weeks to its highest level, which was maintained for at least 3 months following grafting. Along with the elevated blood pressure, the heart weight increased by 80% compared to controls. Behavioral changes were also evident in the G-SHR rats, and these were similar to those of the native SHR strain. In situ hybridization histochemistry showed a 40% elevation in VIP transcripts in the suprachiasmatic nucleus of the host G-SHR brain compared to controls. These studies demonstrate that transplantation of embryonic SHR HTH tissue into brains of adult normotensive rats results in the development of hypertensive characteristics in the host. It thus appears that the HTH is a prime candidate for the source of changes leading to spontaneous hypertension in mammals.

VIP-mRNA is increased in hypertensive rats.

Vasoactive intestinal peptide (VIP) is a potent vasodilator. We therefore set out to investigate VIP-gene expression in spontaneous hypertensive rats. By quantitative in situ hybridization histochemistry as well as by RNA blot hybridization experiments we discovered a significant increase in VIP transcripts in the brains of those hypertensive rats. We suggest that the increase in VIP-gene expression may play a compensatory role in these rats where otherwise the rise in blood pressure may have had a much more adverse effect.