Altered gene expression in hippocampus and depressive-like behavior in young adult female mice by early protein malnutrition.

Perinatal development represents a critical period in the life of an individual. A common cause of poor development is that which comes from undernutrition or malnutrition. In particular, protein deprivation during development has been shown to have deep deleterious effects on brain's growth and plasticity. Early-life stress has also been linked with an increased risk to develop different psychopathologies later in life. We have previously shown that perinatal protein malnutrition in mice leads to the appearance of anxiety-related behaviors in the adulthood. We also found evidence that the female offspring was more susceptible to the development of depression-related behaviors. In the present work, we further investigated this behavior together with its molecular bases. We focused our study on the hippocampus, as it is a structure involved in coping with stressful situations. We found an increase in immobility time in the forced swimming test in perinatally malnourished females, and an alteration in the expression of genes related with neuroplasticity, early growth response 1, calcineurin and c-fos. We also found that perinatal malnutrition causes a reduction in the number of neurons in the hippocampus. This reduction, together with altered gene expression, could be related to the increment in immobility time observed in the forced swimming test.

Male courtship behavior of the South American fruit fly, Anastrepha fraterculus, from an Argentinean laboratory strain.

The South American fruit fly Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) is a pest of fruit species of warm regions of the Americas, including Argentina. Some authors claim that this taxon includes a group of cryptic species. In order to evaluate possible targets of sexual selection, it is necessary to analyze ethological aspects of male courtship and identify particular steps that strongly influence mating success. A mating test designed to evaluate behavioral differences between insects that achieve copulation (successful males) and those that did not mate (unsuccessful males) could also be relevant for the possible implementation of control programs based on sterile insect technique. Reared insects need to be evaluated periodically, since genetic drift and artificial selection associated with rearing conditions could have a detrimental effect on their ability to compete for matings in nature. In this study, courtship behavior of A. fraterculus males from a laboratory strain was analyzed for the first time through video recordings. Three components for the activities were identified: calling, wing positions, and movements. Also, the time that males spent on each step of the courtship was registered, including the last activities before attempting copulation. Data showed that mating achievement occurs relatively quickly; 65% of the successful males reached copulation within the first ten minutes after the male and female were placed together. Behavioral differences were detected between successful and unsuccessful males. The former group tended to invest more time in activities directly related with mating (Spin, Arrowhead, Attempt); however, as courtship progressed, unsuccessful males increased the time dedicated to activities not directly associated to mating (Call 0, Relax,Stationary). There was not a single sequence of activities leading to success, but the analysis of the last activities performed before mating attempts indicated that the most frequent position before successful attempts was Arrowhead, occurring in 68% of cases, whereas in unsuccessful males this position was observed only 18% of the time before mounting. Although the behavior of the strain analyzed here should be compared with that of natural populations, one would not expect to observe significant differences as compatibility and competitiveness with wild collected flies was previously shown under field cage conditions. Behavioral tests such as those applied here might be important to assess quality of mass reared strains for sterile insect technique implementation programs.

Dynamic imaging reveals that brain-derived neurotrophic factor can independently regulate motility and direction of neuroblasts within the rostral migratory stream.

Neuronal precursors generated in the subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Although, the mechanisms regulating this migration remain largely unknown. Studies have shown that molecular factors, such as brain-derived neurotrophic factor (BDNF) emanating from the OB, may function as chemoattractants drawing neuroblasts toward their target. To better understand the role of BDNF in RMS migration, we used an acute slice preparation from early postnatal mice to track the tangential migration of GAD65-GFP labeled RMS neuroblasts with confocal time-lapse imaging. By quantifying the cell dynamics using specific directional and motility criteria, our results showed that removal of the OB did not alter the overall directional trajectory of neuroblasts, but did reduce their motility. This suggested that additional guidance factors present locally within the RMS region also contribute to this migration. Here we report that BDNF and its high affinity receptor, tyrosine kinase receptor type 2 (TrkB), are indeed heterogeneously expressed within the RMS at postnatal day 7. By altering BDNF levels within the entire pathway, we showed that reduced BDNF signaling changes both neuroblast motility and direction, while increased BDNF levels changes only motility. Together these data reveal that during this early postnatal period BDNF plays a complex role in regulating both the motility and direction of RMS flow, and that BDNF comes from sources within the RMS itself, as well as from the olfactory bulb.

Galpha(olf) levels are regulated by receptor usage and control dopamine and adenosine action in the striatum.

In the striatum, dopamine D(1) and adenosine A(2A) receptors stimulate the production of cAMP, which is involved in neuromodulation and long-lasting changes in gene expression and synaptic function. Positive coupling of receptors to adenylyl cyclase can be mediated through the ubiquitous GTP-binding protein Galpha(S) subunit or through the olfactory isoform, Galpha(olf), which predominates in the striatum. In this study, using double in situ hybridization, we show that virtually all striatal efferent neurons, identified by the expression of preproenkephalin A, substance P, or D(1) receptor mRNA, contained high amounts of Galpha(olf) mRNA and undetectable levels of Galpha(s) mRNA. In contrast, the large cholinergic interneurons contained both Galpha(olf) and Galpha(s) transcripts. To assess the functional relationship between dopamine or adenosine receptors and G-proteins, we examined G-protein levels in the striatum of D(1) and A(2A) receptor knock-out mice. A selective increase in Galpha(olf) protein was observed in these animals, without change in mRNA levels. Conversely, Galpha(olf) levels were decreased in animals lacking a functional dopamine transporter. These results indicate that Galpha(olf) protein levels are regulated through D(1) and A(2A) receptor usage. To determine the functional consequences of changes in Galpha(olf) levels, we used heterozygous Galpha(olf) knock-out mice, which possess half of the normal Galpha(olf) levels. In these animals, the locomotor effects of amphetamine and caffeine, two psychostimulant drugs that affect dopamine and adenosine signaling, respectively, were markedly reduced. Together, these results identify Galpha(olf) as a critical and regulated component of both dopamine and adenosine signaling.

Symmetry, stereotypy, and topography of odorant representations in mouse olfactory bulbs.

The molecular basis of vertebrate odorant representations has been derived extensively from mice. The functional correlates of these molecular features were visualized using optical imaging of intrinsic signals in mouse olfactory bulbs. Single odorants activated clusters of glomeruli in consistent, restricted portions of the bulb. Patterns of activated glomeruli were clearly bilaterally symmetric and consistent in different individual mice, but the precise number, position, and intensity of activated glomeruli in the two bulbs of the same individual and between individuals varied considerably. Representations of aliphatic aldehydes of different carbon chain length shifted systematically along a rostral-caudal strip of the dorsal bulb, indicating a functional topography of odorant representations. Binary mixtures of individual aldehydes elicited patterns of glomerular activation that were topographic combinations of the maps for each individual odor. Thus the principles derived from the molecular organization of a small subset of murine olfactory receptor neuron projection patterns-bilateral symmetry, local clustering, and local variability-are reliable guides to the initial functional representation of odorant molecules.

G(olf)alpha mediates dopamine D1 receptor signaling.

It is generally assumed that the coupling of dopamine D1 receptors to adenylyl cyclase is mediated by the stimulatory GTP-binding protein G(s). However, the striatum contains little G(s)alpha subunit, whereas it expresses high levels of G(olf)alpha, a close relative of G(s)alpha that is also expressed in olfactory receptor neurons. We used G(olf)alpha knockout mice to examine the functional coupling of D1 receptors. We found that these mice showed no hyperlocomotor response to either the D1 agonist SKF-81297 or the psychostimulant cocaine. Moreover, G(olf)alpha knockout mice did not display cocaine-induced c-fos expression in the striatum. Finally, in the absence of G(olf)alpha, striatal D1 receptors have a decreased affinity for dopamine. Thus coupling to G(olf)alpha appears to mediate D1 signaling in the striatum.

A map of pheromone receptor activation in the mammalian brain.

In mammals, the detection of pheromones is mediated by the vomeronasal system. We have employed gene targeting to visualize the pattern of projections of axons from vomeronasal sensory neurons in the accessory olfactory bulb. Neurons expressing a specific receptor project to multiple glomeruli that reside within spatially restricted domains. The formation of this sensory map in the accessory olfactory bulb and the survival of vomeronasal organ sensory neurons require the expression of pheromone receptors. In addition, we observe individual glomeruli in the accessory olfactory bulb that receive input from more than one type of sensory neuron. These observations indicate that the organization of the vomeronasal sensory afferents is dramatically different from that of the main olfactory system, and these differences have important implications for the logic of olfactory coding in the vomeronasal organ.

Mice deficient in G(olf) are anosmic.

We have used gene targeting to examine the role of the G alpha subunit, G(olf), in olfactory signal transduction. Mice homozygous for a null mutation in G(olf) show a striking reduction in the electrophysiological response of primary olfactory sensory neurons to a wide variety of odors. Despite this profound diminution in response to odors, the topographic map of primary sensory projections to the olfactory bulb remains unaltered in G(olf) mutants. Greater than 75% of the G(olf) mutant mice are unable to nurse and die within 2 days after birth. Rare surviving homozygotes mate and are fertile, but mutant females exhibit inadequate maternal behaviors. Surviving homozygous mutant mice also exhibit hyperactive behaviors. These behavioral phenotypes, taken together with the patterns of G(olf) expression, suggest that G(olf) is required for olfactory signal transduction and may also function as an essential signaling molecule more centrally in the brain.

Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity.

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 (NT-3) are the three members of the neurotrophin family known to exist in mammals. Recently, a fourth neurotrophin (designated neurotrophin-4 or NT-4), which shares all of the features found in the mammalian neurotrophins, has been identified in Xenopus and viper. We used sequences specific to the Xenopus/viper NT-4 to isolate a neurotrophin from both human and rat genomic DNA that appears to represent the mammalian counterpart of Xenopus/viper NT-4. Human NT-4 as well as a human NT-4 pseudogene colocalize to chromosome 19 band q13.3. Mammalian NT-4 has many unusual features compared to the previously identified neurotrophins and is less conserved evolutionarily than the other neurotrophins. However, mammalian NT-4 displays bioactivity and trk receptor specificity similar to that of Xenopus NT-4.

Gene sequences of chicken BDNF and NT-3.

The respective amino acid sequences of mature brain-derived neurotrophic factor (BDNF) and of mature neurotrophin-3 (NT-3) are identical among mammals, making these among the structurally conserved factors known. Here we show that only a single conservative amino acid substitution distinguishes the chicken mature NT-3 protein from its mammalian counterpart. Chicken mature BDNF shows slightly more variation, differing from mammalian BDNF at several positions. We also note the presence of amino acid sequence motifs in the precursor protein sequences of chicken BDNF and NT-3 that are universally conserved among all known mammalian neurotrophin precursors and have been demonstrated to play a crucial role in promoting correct processing of the pro-proteins.

Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions, and chromosomal localizations.

The development and maintenance of the vertebrate nervous system depends upon neuronal survival proteins known as neurotrophic factors. Nerve growth factor (NGF) remains the best characterized neurotrophic molecule. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are two recently cloned neurotrophic factors that are homologous to NGF. Here we describe the molecular cloning of the human and rat genes encoding BDNF, as well as the isolation of the human NT-3 gene. On the basis of comparison of our genomic and cDNA clones with those of previously isolated BDNF and NT-3 genes and cDNAs, we make inferences about the structures of processed transcripts derived from the neurotrophin genes and the protein precursors they encode. We demonstrate that the mature form of BDNF is identical in all mammals examined, and that the same is true of the mature form of NT-3. Furthermore, the respective tissue-distributions and neuronal specificities of NT-3 and BDNF are also conserved among mammals. Finally, we localize the gene encoding human BDNF (gene symbol designated BDNF) to chromosome 11, band p13, and the gene encoding human NT-3 (gene symbol designated NTF3) to chromosome 12, band p13.

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.

Neurotrophin-3: a neurotrophic factor related to NGF and BDNF.

The development and maintenance of the nervous system depends on proteins known as neurotrophic factors. Although the prototypical neurotrophic factor, nerve growth factor (NGF), has been intensively studied for decades, the discovery and characterization of additional such factors has been impeded by their low abundance. Sequence homologies between NGF and the recently cloned brain-derived neurotrophic factor (BDNF) were used to design a strategy that has now resulted in the cloning of a gene encoding a novel neurotrophic factor, termed neurotrophin-3 (NT-3). The distribution of NT-3 messenger RNA and its biological activity on a variety of neuronal populations clearly distinguish NT-3 from NGF and BDNF, and provide compelling evidence that NT-3 is an authentic neurotrophic factor that has its own characteristic role in vivo.

Neurotrophic factors, their receptors, and the signal transduction pathways they activate.

Our studies of the spatiotemporal availability of neurotrophic factors, coupled with tagged ligand binding assays that identify cell bearing receptors for these factors, should lead toward defining the physiological roles of these molecules in the animal. The use of the tagged ligands to identify factor-responsive cell lines has also provided new model systems for the examination of ligand-receptor interactions, as well as for the study of the subsequent induction of intracellular response pathways. To obtain insights into such intracellular pathways, we have molecularly cloned genes encoding a family of serine-threonine protein kinases, most closely related to kinases involved in the yeast response to pheromones. These kinases may be crucial regulators of early steps in the response of mammalian cells to neurotrophic factors as well as other extracellular signals.