Anatomical Phenotypes Research Articles

CTL priming by CD8(+) and CD8(-) dendritic cells in vivo.

Two distinct developmental pathways are driving the formation of myeloid- and lymphoid-related dendritic cells (DC) which differ in anatomical localization and phenotype. In terms of function, it has been hypothesized that only the myeloid-related CD8(-) DC are able to initiate immune responses, whereas the lymphoid-related CD8(+) DC have been suggested to induce tolerance. Here we show that both subsets activate CD8(+) T cells in vitro and induce protective anti-viral CTL responses in vivo. Thus, vaccine strategies using peptide-pulsed DC do not have to take into account DC subsets for priming.

Distribution and phenotype of neurons containing the ATP-sensitive K + channel in rat brain

Select groups of neurons within the brain alter their firing rate when ambient glucose levels change. These glucose-responsive neurons are integrated into systems which control energy balance in the body. They contain an ATP-sensitive K + channel (K ATP) which mediates this response. K ATP channels are composed of an inwardly rectifying pore-forming unit (Kir 6.1 or Kir 6.2) and a sulfonylurea binding site. Here, we examined the anatomical distribution and phenotype of cells containing Kir 6.2 mRNA within the rat brain by combinations of in situ hybridization and immunocytochemistry. Cells containing Kir 6.2 mRNA were widely distributed throughout the brain without apparent concentration in areas known to contain specific glucose-responsive neurons. Kir 6.2 mRNA was present in neurons expressing neuron-specific enolase, tyrosine hydroxylase, neuropeptide Y (NPY) and the glutamic acid decarboxylase isoform, GAD 65. No astrocytes expressing glial fibrillary acidic protein or oligodendrocytes expressing carbonic anhydrase II were found to co-express Kir 6.2 mRNA. Virtually all of the NPY neurons in the hypothalamic arcuate n. and catecholamine neurons in the substantia nigra, pars compacta and locus coeruleus contained Kir 6.2 mRNA. Epinephrine neurons in the C2 area also expressed high levels of Kir 6.2, while noradrenergic neurons in A5 and A2 areas expressed lower levels. The widespread distribution of Kir 6.2 mRNA suggests that the K ATP channel may serve a neuroprotective role in neurons which are not directly involved in integrating signals related to the body's energy homeostasis.

Transgenic mice transcribing the human H1 zero histone gene exhibit a normal phenotype.

The linker histone H1degree accumulates in terminally differentiating cells and replaces other members of the H1 histone family, even in the absence of cell division. To study the role of H1degree in vivo, we have created two lines of transgenic mice with either the human H1degree promoter (HH minigene) or the mouse metallothionein T promoter (MH minigene) upstream of the human H1degree gene. Mice bearing the minigenes HH or MH overexpress human H1degree mRNA at 10-20-fold higher levels than in normal mice in a constitutive or metal-inducible manner. In contrast to this increase in mRNA content, which was studied in liver, kidney and brain, no significant changes in the relative proportions of the H1 protein subtypes, including H1degree were observed. Transgenic mice exhibited normal anatomic phenotypes, growth rates and reproduction rates. Thus, our results suggest a posttranscriptional and/or translational mechanism that compensates the unbalanced linker-histone expression in different tissues.

RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice.

Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.

Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm).

Mutations in 12 genes regulating Drosophila melanogaster mushroom body (MB) development were each studied in two genetic backgrounds. In all cases, brain structure was qualitatively or quantitatively different after replacement of the "original" genetic background with that of the Canton Special wild-type strain. The mushroom body miniature gene (mbm) was investigated in detail. mbm supports the maintenance of MB Kenyon cell fibers in third instar larvae and their regrowth during metamorphosis. Adult mbm1 mutant females are lacking many or most Kenyon cell fibers and are impaired in MB-mediated associative odor learning. We show here that structural defects in mbm1 are apparent only in combination with an X-linked, dosage-dependent modifier (or modifiers). In the Canton Special genetic background, the mbm1 anatomical phenotype is suppressed, and MBs develop to a normal size. However, the olfactory learning phenotype is not fully restored, suggesting that submicroscopic defects persist in the MBs. Mutant mbm1 flies with full-sized MBs have normal retention but show a specific acquisition deficit that cannot be attributed to reductions in odor avoidance, shock reactivity, or locomotor behavior. We propose that polymorphic gene interactions (in addition to ontogenetic factors) determine MB size and, concomitantly, the ability to recognize and learn odors.

Review : Single-Cell mRNA Amplification: Implications for Basic and Clinical Neuroscience

The application of molecular biological techniques to the study of single cells has provided a unique window for exploring the mechanisms that underlie integrated cellular function. Analysis of gene expression in indi vidual cells of the central nervous system is critical to understanding how distinct cell populations with differing chemical and anatomic phenotypes respond to pharmacological agents or are altered in disease states. Quantification of mRNA by single-cell analysis gives a high-resolution picture of changes in gene expression within individual cells, whereas more conventional types of mRNA analysis may obscure subtle changes in gene expression because of a lack of change in surrounding cells that are included in the mRNA sample. In addition, the sensitivity for detecting low levels of mRNA is enhanced when individual versus groups of cells are analyzed. With the advent of various mRNA amplification strategies, it is now possible to determine the mRNA composition or "expression profile" of individual cells. Information about relative levels of different mRNAs, the subcellular localization of mRNAs, and insight into cell-specific RNA splicing and RNA editing can be obtained. When these molecular data are combined with electrophysiological, morpho logical, immunohistochemical, and anatomical analyses, a detailed portrait of neuronal functioning can be obtained. Moreover, alterations in cellular functioning induced by physiological manipulation, drug adminis tration, or disease state can be monitored by combining these approaches. This precise cellular information may be useful in developing pharmaceuticals designed to alter mRNA levels or protein levels in a predictable manner (transcript-aided drug design) to elicit specific physiological states. The Neuroscientist 1:200-211, 1995

Transplantation of mesencephalic cell suspensions from wild-type and heterozygous weaver mice into the denervated striatum: Assessing the role of graft-derived dopaminergic dendrites in the recovery of function

The Weaver ( wv) mutation leads to a loss of mesencephalic dopamine cells and nigrostriatal dopamine axons in homozygosity ( wv/wv) and to a deficiency of nigral dopaminergic dendrites without a concomitant loss of dopamine cell somata or axons in heterozygosity ( wv/+). Previous studies have shown that grafts of foetal dopamine cells from wild-type (+/+) donors can survive when implanted into the wv/wv striatum, supply both an axonal and a dendritic innervation to the host, establish synaptic connections with host striatal neurons, and bring about a functional recovery evidenced by rotational asymmetry tests. The aims of the present study were to examine whether wv/+ dopamine cells maintain a “dendrite-poor” phenotype after transplantation to the denervated striatum, and to compare their functional effects with those of wild-type (+/+) grafts in reversing amphetamine-induced turning behaviour. To that end, +/+ and wv/+ ventral mesencephalic tissue (dissected out from E10-E12 foetal mice and made into a cell suspension by enzymatic and mechanical dissociation) was stereotactically grafted into the right striatum of either wv/wv hosts or +/+ hosts subjected in advance to 6-OHDA lesions of the right substantia nigra. Viability and morphology of grafted neurons were assessed by tyrosine hydroxylase immunocytochemistry on serial sections of the host forebrains. Dopamine cell bodies survived in comparable numbers in the grafts regardless of donor genotype; however, grafts of either genotype contained fewer dopaminergic cells when they were hosted in the wv/wv striatum as compared to the striatum of +/+ mice with 6-OHDA lesions. Despite the survival of cell somata, the dendritic arborisation of wv/+ cells was strikingly poorer than that of +/+ cells in grafts placed into both host types, most likely reflecting their in situ phenotypic abnormality. Recipient wv/wv mice with +/+ and wv/+ grafts exhibited 88% and 83% left rotations, respectively; 6-OHDA hosts with +/+ and wv/+ grafts showed 178% and 165% reversals of asymmetry, respectively. The differences between the effects of +/+ and wv/+ grafts were not statistically significant. We conclude that (i) wv/+ and +/+ dopamine cell somata survive in comparable numbers after intrastriatal grafting; (ii) grafted wv/+ dopamine cells express an anatomical phenotype consistent with that seen in the wv/+ substantia nigra in situ; and (iii) the axonal innervation supplied by wv/+ grafts to the denervated striatum induces a functional recovery comparable to that brought about by +/+ cells, which in addition supply a substantial dendritic innervation to the host; (iv) the wv/wv host environment may be associated with smaller numbers of graft dopamine neurons compared to the environment of +/+ mouse hosts with 6-OHDA lesions.

Transplantation of mesencephalic cell suspensions from wild-type and heterozygous Weaver mice into the denervated striatum: assessing the role of graft-derived dopaminergic dendrites in the recovery of function.

The Weaver (wv) mutation leads to a loss of mesencephalic dopamine cells and nigrostriatal dopamine axons in homozygosity (wv/wv) and to a deficiency of nigral dopaminergic dendrites without a concomitant loss of dopamine cell somata or axons in heterozygosity (wv/+). Previous studies have shown that grafts of foetal dopamine cells from wild-type (+/+) donors can survive when implanted into the wv/wv striatum, supply both an axonal and a dendritic innervation to the host, establish synaptic connections with host striatal neurons, and bring about a functional recovery evidenced by rotational asymmetry tests. The aims of the present study were to examine whether wv/+ dopamine cells maintain a "dendrite-poor" phenotype after transplantation to the denervated striatum, and to compare their functional effects with those of wild-type (+/+) grafts in reversing amphetamine-induced turning behaviour. To that end, +/+ and wv/+ ventral mesencephalic tissue (dissected out from E10-E12 foetal mice and made into a cell suspension by enzymatic and mechanical dissociation) was stereotactically grafted into the right striatum of either wv/wv hosts or +/+ hosts subjected in advance to 6-OHDA lesions of the right substantia nigra. Viability and morphology of grafted neurons were assessed by tyrosine hydroxylase immunocytochemistry on serial sections of the host forebrains. Dopamine cell bodies survived in comparable numbers in the grafts regardless of donor genotype; however, grafts of either genotype contained fewer dopaminergic cells when they were hosted in the wv/wv striatum as compared to the striatum of +/+ mice with 6-OHDA lesions. Despite the survival of cell somata, the dendritic arborisation of wv/+ cells was strikingly poorer than that of +/+ cells in grafts placed into both host types, most likely reflecting their in situ phenotypic abnormality. Recipient wv/wv mice with +/+ and wv/+ grafts exhibited 88% and 83% left rotations, respectively; 6-OHDA hosts with +/+ and wv/+ grafts showed 178% and 165% reversals of asymmetry, respectively. The differences between the effects of +/+ and wv/+ grafts were not statistically significant.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of the c-fos Gene in Prodynorphin- and Proenkephalin-Expressing Cells of Nucleus Tractus Solitarius after Seizures

We performed studies to determine the anatomical regions and chemical phenotypes of neurons within the rat medulla oblongata activated by pentylenetetrazole-induced seizures. Activated cells were identified by their expression of the c-fos gene, detected by in situ hybridization for c-fos mRNA and immunocytochemistry for Fos protein. Activated cells were located predominantly in nucleus tractus solitarius (NTS), with c-fos mRNA appearing within 20 min after seizures (peak at 1-2 h), followed by Fos immunoreactivity visible at 1 h (peak at 2-4 h). Neither nonspecific noxious stimulation by intraperitoneal injection of saline nor brief exposure to hypoxic or hypercapnic gas mixtures to stimulate chemoreceptors reproduced this pattern of labeling. Prodynorphin or proenkephalin mRNA, detected by in situ hybridization, was colocalized with Fos immunoreactivity in many NTS cells. Thus, seizures activate neuronal pathways in the medulla oblongata which express genes for endogenous opioids. Potential long-term effects of seizures are suggested by the in situ hybridization finding that NTS prodynorphin mRNA increased 24 h after seizures compared to control levels.

Teratology, Genetics, and Recurrence Risks

This brief review has described historic highlights of etiologic knowledge, current concepts in the categorization of cardiovascular anomalies based upon ongoing advances in teratology, and epidemiologic evaluations of biologic and xenobiotic risk factors with emphasis on the teratogenic roles of maternal diabetes, hyperphenylalaninemia, and parental exposures to alcohol, drugs, solvents, pesticides, lead, and other toxic substances. Evidence is presented for a strong genetic basis of cardiovascular maldevelopment requiring further studies to define at-risk families. Counseling and personal and societal preventive interventions may reduce the occurrence of some forms of CHD.

Unique cerebellar phenotype combining granule and Purkinje cell loss: Morphological evidence forweaver *pcd double mutant mice

Weaver (wv/wv) mutant mice lose most granule cells of the cerebellum during the first 2 weeks of postnatal life; 'Purkinje cell degeneration' (pcd/pcd) mutants lose virtually all Purkinje cells between postnatal days 17 and 45. Both these neurological mutations are autosomal recessive. We designed a breeding protocol that, in theory, should result in the production of mice with a doubly mutant, wv/wv*pcd/pcd, genotype. Some of the offspring of such crosses had a novel cerebellar phenotype in which both granule and Purkinje cells underwent degeneration, leading to a highly atrophic cortex. This phenotype is what would be expected in wv/wv*pcd/pcd double mutants, and the proportion of such progeny obtained fits with genetic expectations. We propose that (1) wv/wv*pcd/pcd double mutant mice are viable, and (2) the anatomical phenotype of such mice is a combined expression of the component phenotypes.

Androgen and Estrogen Synthesis in the Fetal Guinea Pig Gonad12

Bisected fetal guinea pig ovaries and testes were examined in vitro for their ability to synthesize androgens from [1-’ CI sodium acetate and estrogens from either I i-� HI androstenedione or 11 ,2-� HI androstenedione. With [1-’ � Cl sodium acetate as the substrate, hexane-soluble steroids, androstenedione and testosterone were synthesized by fetal testes from Day 30 to 65 of development. To a lesser extent, ovaries also synthesized hexane-soluble steroids, but only when ovaries from various stages of gestation were pooled was the synthesis of ‘4C-androstenedione detected. Radioactive testosterone was not detected in ovarian incubations and at no time was 4C-dihydrotestosterone produced by fetal ovaries or testes. More radioactive testosterone and androstenedione (d/min X mg tissue) appeared in Day 30 testicular incubations than in incubations of organs from later stages of development. In addition, between Days 30 and 50 of gestation more testosterone than androstenedione was recovered from the medium, but by Day 60-65 androstenedione predominated. Total activity (d/min X gonad) generally paralleled organ weight and increased with advancing gestation. The rate of 3H20 formation was utilized as an index of 1i-� HI androstenedione aromatization. Ovaries between 30 and 65 days of gestation were considerably more active (d/min X mg tissue or d/min X gonad) in synthesizing 3H20 (2.3-0.3% of the radioactive substrate) than corresponding testes in which activity was not significantly different than that noted for abdominal muscle. When ovaries were incubated with l1,2-� I androstenedione for 60 mm, the rate of synthesis of H2 0, 3H-estrone and 3H-estradiol-17� was constant. In this case tritiated estrogens accounted for 16-23% of the total product radioactivity. The results indicate that fetal guinea pig testes and ovaries contain the necessary enzymes for de novo androgen and estrogen synthesis at a time during development when anatomical and behavioral phenotypes are differentiated.